With a now-rescinded White House directive that threw millions of federal dollars for education and research into uncertainty, President Trump and his allies tried to prove they were not bluffing with their campaign threats to target universities.

But before President Trump even returned to office, many of the nation’s well-known universities were already preparing to fight back.

While few college presidents are especially eager to spar with Mr. Trump and Vice President JD Vance in public, schools have been marshaling behind-the-scenes counteroffensives against promises of an onslaught of taxes, funding cuts and regulations.

Some universities have hired powerhouse Republican lobbying firms. Others are strengthening, or rebuilding, their presences in Washington.

Many are quietly tweaking their messaging and policies, hoping to deter policymakers who know it can be good politics to attack higher education — even when they themselves are products of the schools they castigate on cable television. Rutgers University, for example, announced last week that it would cancel a conference on diversity, equity and inclusion, a focus of the new administration.

A spokeswoman for the university said the decision, which prompted criticism, was made after many speakers from a federally funded program withdrew from the conference, citing an executive order that targets the topic.

“There’s a concern among a lot of campuses,” said Kenneth K. Wong, a professor of education policy at Brown.

Some efforts to rehabilitate higher education’s reputation were already in the works, a response to attacks leaders in Congress made after campus protests over the war in Gaza. But now university officials are confronting an administration whose leaders have made clear their contempt for some wings of higher education. Mr. Trump has said schools are dominated by “Marxists, maniacs and lunatics,” and Mr. Vance has called them “insane.”

The ominous saber rattling from Mr. Trump and his allies includes threats to endowments, federal research funding, student financial aid, diversity initiatives and the potential deportation of roughly 400,000 undocumented students enrolled in U.S. schools.

Several major universities have responded by hiring lobbyists whom Republican leaders might view favorably. Harvard University has turned to a Capitol Hill heavyweight, Ballard Partners, the former firm of both Mr. Trump’s attorney general-designate, Pam Bondi, and his chief of staff, Susie Wiles. Columbia University signed up with BGR Government Affairs, which counts Haley Barbour, a former Mississippi governor and Republican National Committee chairman, among its co-founders.

Duke University, which has an in-house government relations effort, brought in DLA Piper as an adviser. One of the firm’s executives is Richard Burr, a Republican who represented North Carolina (where Duke is located) in the Senate for 18 years.

The University of Notre Dame recently registered its own lobbyists for the first time since Mr. Trump’s previous term. And Yale University is beginning its own theater of operations in Washington.

“The university decided to open an office in Washington, D.C. after conducting benchmarking among peer institutions,” Karen Peart, a Yale spokeswoman, wrote in an email, citing upcoming higher education “issues” on Capitol Hill.

The latest activity in Washington came after some other schools ramped up lobbying efforts.

As recently as 2022, Washington University in St. Louis paid $50,000 for its lobbying in the capital. The next year, it raised that spending to $250,000. That exploded to $720,000 in 2024, federal records show. A university spokeswoman did not comment.

Across the country, university officials and their allies said that they were somewhat more prepared for what to expect under Mr. Trump than they were when he first ascended to power in 2017. Eight years later, they said, they had a better sense of Mr. Trump’s approach to the presidency and have also looked for insights into his administration’s ambitions in the “Project 2025” plan, which is closely linked to many of his appointees.

The administration wasted no time in launching those plans with a flurry of executive orders in its first week. One seeks to ban diversity, equity and inclusion initiatives, including those run by contractors that receive federal student aid funding — a category that includes virtually every campus.

Mr. Trump also ordered federal agencies to compile lists of “nine potential civil compliance investigations” of organizations, including higher-education institutions with endowments over $1 billion.

In a public conference call on Monday sponsored by DLA Piper, Mr. Burr said that while the rest of the Trump administration’s higher education policy was not yet entirely clear, “we believe that endowments are a target of revenue, potentially, in a tax bill.”

Few topics are as alarming to the leaders of the country’s wealthiest universities.

Endowments were largely exempt from taxation for years. But in 2017, during Mr. Trump’s first term, Republicans led a charge to impose a 1.4 percent excise tax on the investment income of large private university endowments. Now there are discussions of raising it to 14 percent, or even 21 percent.

As a senator, Mr. Vance was a leading proponent of increasing the endowment tax, proposing an increase to 35 percent for endowments of $10 billion or more. Despite his Yale law degree, funded partly by the university, Mr. Vance has previously called for an “attack” on universities.

“Why is it that we allow these massive hedge funds pretending to be universities to enjoy lower tax rates than most of our citizens, people who are struggling to put food on the table?” he said when he was a senator, adding: “It’s insane. It’s unfair.”

At least 56 schools were forced to pay the 1.4 percent tax in 2023, totaling more than $380 million, according to an analysis by the National Association of College and University Business Officers. Records show that representatives of major universities were busy presenting their anti-endowment positions on Capitol Hill last year. In the fourth quarter, about 10 top schools, including Stanford and Cornell, lobbied on the tax.

They have often built their case around what they contend would be lost if universities had to pay more of the government’s bills: money that they use for research and tuition support, particularly for low-income students.

At Wesleyan University, for example, that amounted to $85 million last year that served 1,500 students, according to Michael S. Roth, Wesleyan’s president.

“So it’s real money,” Dr. Roth said, adding that a tax increase would make it harder for the university to support students. He added, “It means we will be serving fewer worthy applicants.” Dr. Roth said that Wesleyan would not be hiring outside lobbyists but, instead, would use that money to assist students.

Mr. Burr also said universities would be affected if the Trump administration targeted funds for research. He noted that the National Institutes of Health and the Centers for Disease Control and Prevention had both recently issued directives to suspend public communications, research-grant reviews, travel and training for scientists.

On Monday evening, the administration also issued a sweeping pause on trillions in federal grant funding, which a federal judge blocked about 24 hours later — but only after a day of chaos and tumult for campus leaders.

Ted Mitchell, the president of the American Council on Education, which represents more than 1,600 campuses nationwide, called it the “most irresponsible public policy” he had ever witnessed. The organization called for the order’s reversal; the White House backed away from the order on Wednesday.

The pause had been designed to give the administration time to determine whether grants align with Mr. Trump’s priorities. In the 2023 fiscal year, universities received close to $60 billion in federal funding for research.

Barbara Snyder, the president of the Association of American Universities, which includes dozens of the most prominent schools in the country, noted that the explosion of anger in Washington toward universities was not necessarily new.

“It’s more challenging than it was 20 years ago,” she said, but added: “I don’t think this has all been an overnight change.”

Even as universities muster defenses, no consensus has emerged among them about how best to approach the second iteration of Mr. Trump’s Washington.

“Our institutions,” Ms. Snyder said, “have their own ways of doing these things.”