On Monday, Gov. Ron DeSantis of Florida went out on a limb. On Tuesday, it snapped.

A day after he swooped into the Republican primary for Kentucky governor with a last-minute endorsement — a move that turned the race into an obvious proxy fight between himself and former President Donald J. Trump — Mr. DeSantis watched his chosen candidate lose in a landslide to the Trump-backed rival.

To make matters worse for Mr. DeSantis, a Republican he had endorsed conceded to a Democratic opponent in the mayor’s race in Jacksonville, the largest city in his state.

Mr. DeSantis’s preparations to enter the 2024 primary are intensifying. He has held a series of private dinners in Tallahassee with top donors, and on Tuesday he took a direct shot at Mr. Trump over his dodging whether he would sign a six-week abortion ban.

But on Monday, Mr. DeSantis made a last-minute endorsement and robocall for Kelly Craft, a former United Nations ambassador under Mr. Trump and a member of a Republican megadonor family.

The move confounded Kentucky Republicans and those working for her rivals: While Ms. Craft spent heavily on the race, polls had suggested she was headed for defeat to Daniel Cameron, the state’s attorney general, an ally of Senator Mitch McConnell who had garnered Mr. Trump’s endorsement in June 2022. Representatives for Mr. DeSantis declined to comment.

“Kelly shares the same vision we do in Florida,” Mr. DeSantis said in a recording that was sent to Republican voters on the eve of the primary.

It ended up being far from close. With nearly 90 percent of ballots counted, she was in a distant third, earning just 17 percent of the vote to Mr. Cameron’s 47 percent.

“Let me just say,” Mr. Cameron said in his victory speech, “the Trump culture of winning is alive and well in Kentucky!”

His choice of words was telling: As Mr. DeSantis nears the announcement of a presidential campaign, his stump speech has often called on the Republican Party to end its “culture of losing” during the Trump era. On Monday, the phrase was splashed across the front page of The Des Moines Register after the governor campaigned in Iowa over the weekend.

The Trump team cheered Mr. Cameron’s line. In fact, one of Mr. Trump’s top advisers, Chris LaCivita, had presaged it less than an hour before Mr. Cameron spoke. When the race was called, Mr. LaCivita wrote on Twitter, “so much for the #alwaysbackdown culture of winning.”

Never Back Down is the name of the main super PAC backing Mr. DeSantis. One of that super PAC’s top strategists is Jeff Roe, whose consulting firm also worked for Ms. Craft.

The unsuccessful election-eve endorsement of Ms. Craft was similar to the last-minute backing that Mr. DeSantis gave to Harmeet Dhillon in the race to lead the Republican National Committee in January.

Mr. DeSantis called for “new blood” the day before that vote. The incumbent, Ronna McDaniel, won easily the next day.

Meanwhile, Mr. DeSantis’s night did not get better in Jacksonville, where Daniel Davis, the Republican endorsed by the governor, lost to Donna Deegan, a Democrat, for an open seat. Mr. DeSantis had provided little support to Mr. Davis beyond his endorsement, not visiting the city to campaign. Early results showed Ms. Deegan leading Mr. Davis with roughly 52 percent of the vote.

Jacksonville has had Republican mayors for most of the last 30 years.