For weeks, he's resisted taking on Donald Trump, who has eviscerated other Republicans who threatened his status as the front-runner.
That reluctance evaporated during the final GOP debate before "Super Tuesday" as Rubio delivered blow after blow at Trump, attacking him on his immigration, foreign policy and health care positions, along with his business hiring practices.
"You're the only person on this stage that's ever been fined for hiring people to work on your project illegally," Rubio told Trump at the debate that aired on CNN.
Rubio and Trump spoke over each other as the senator accused the real estate mogul of hiring Polish workers. Trump dismissed the episode as something that happened decades ago.
"I guess there's a statute of limitations on lies," Rubio said.
Rubio later mocked Trump for having a "fake school," referring to lawsuits aimed at Trump University alleging fraud and deception.
Trump hit back: "Here's a guy that buys a house for $179,000 -- he sells it to a lobbyist who's probably here for 380,000."
Rubio responded by calling out Trump for his privileged background.
"If he hadn't inherited $200 million, you know where Donald Trump would be right now? Selling watches in Manhattan," Rubio said.
And Rubio challenged Trump on foreign policy.
Trump repeatedly stated his support for Israel, billing himself the most "pro-Israel" candidate in the GOP field. But even as he said he was "totally pro-Israel," he also said he didn't believe there was any reason for labeling Israel and the Palestinians as the "good guy" and the "bad guy."
"The position you've taken is an anti-Israel position," Rubio said.
When Trump said he was simply a "negotiator," Rubio shot back: "The Palestinians are not a real estate deal, Donald."
The tough talk comes as Trump heads into the March 1 Super Tuesday contests with remarkable momentum, after consecutive victories in New Hampshire, South Carolina and Nevada.
Rubio has struggled to step out of Trump's shadow and Thursday's debate could mark a pivotal moment as he seeks to convince voters that he is best positioned to be an alternative to Trump and to unite a fractured Republican Party.
One of the most explosive exchanges between Trump and Rubio came when the candidates were asked to discuss their health care plans.
Trump said he would get rid of barriers between the states --- something that he argued would increase competition. Rubio repeatedly pressed his rival for more details. "What else is a part of your health care plan?"
"When you get rid of the lines, that brings in competition," Trump said.
"Now he's repeating himself," Rubio responded.
"I don't repeat myself. Here's the guy who repeats himself," Trump shot back, referring to Rubio. "I watched him repeat himself five times four weeks ago."
Rubio, appearing amused, quipped: "I saw you repeat yourself five times five seconds ago!"
The senator also accused Trump of reusing the same general points on the campaign trail: "Everyone's dumb, he's going to make America great again, he's gonna win win win, he's winning in the polls."
CNN's Dana Bash cut in to ask whether Trump has anymore to add to his previously stated proposal on health-care reform.
"No, there's nothing to add. What's to add?" Trump said.
Ted Cruz also went on the offensive, saying Trump had previously donated to Democrats who had worked on the so-called Gang of Eight immigration reform bill. Voters should judge a candidate by looking at their "record before they were a candidate for president," Cruz added.
The conservative firebrand senator came in first-place in the Iowa caucuses but has not been able to notch another win since, and has been forthright about the ramifications of Super Tuesday, especially the significance of his home state of Texas, where there are 155 delegates at stake.
A Monmouth University poll released Thursday put Cruz 15 points ahead of Trump in Texas.
Mitt Romney, the 2012 GOP presidential nominee, also played a role in the debate. He sparked controversy Wednesday when he suggested Trump's tax returns could contain a potential "bombshell" and urged the businessman to release his information.
On the debate stage, Trump claimed that he was currently being audited and that he would not release the returns until that process was over.
"I can't release it while I'm under an audit," he said.
Cruz echoed Romney's suggestion that there may be something problematic lurking in Trump's tax returns.
"Donald Trump says he's being audited. I would think that would underscore the need to release those returns," he said. "He doesn't want to do it because presumably there is something in there that's bad."
Cruz and Rubio said they would release new tax returns within the next two days.
Cruz and Rubio also tussled with Trump over replacing Supreme Court Justice Antonin Scalia.
When Cruz vowed to nominate principled constitutionalists to the bench as president, Trump shot back: "Justice (John) Roberts was strongly recommended and pushed by Ted."
"Justice Roberts gave us Obamacare. Might as well be called Robertscare," Trump said. "That is a rough thing and I know Ted feels badly about it."
Cruz slammed Trump for donating to Democrats in the past --- a sign, Cruz warned, that Trump would get to Washington and "cut a deal" on Supreme Court appointees.
Trump responded by blasting Cruz for being an obstructionist in Congress --- "You have to have somebody who can make deals," he said.
Rubio also joined in, saying he doubted Trump would "replace Justice Scalia with someone just like Justice Scalia."
Trump also responded to former Mexican President Vicente Fox's recent response to Trump's claim that Mexico would pay for his proposed U.S.-Mexico wall.
"I'm not going to pay for that f***ing wall. He should pay for it," Fox told Fusion's Jorge Ramos in an interview published Thursday.
On the CNN debate stage, Trump quipped: "The wall just got 10 feet taller."
"I saw him make the statement. I saw him use the word that he used," Trump added. "This guy used a filthy, disgusting word on television ... He should apologize."
Trump went into the debate facing calls to release his tax returns. Mitt Romney told Fox News on Wednesday that there could be a "bombshell" in Trump's tax returns, a line that signaled anxiety inside the GOP establishment that the billionaire businessman might become the nominee.
Trump struck back in a series of tweets and told CNN's Anderson Cooper, "there is no bombshell at all other than I pay a lot of tax and the government wastes the money."