Pence said in a letter that he did not believe, as Trump has claimed, that a vice president has the power to reject some Electoral College votes for a candidate.
"It is my considered judgment that my oath to support and defend the Constitution constrains me from claiming unilateral authority to determine which electoral votes should be counted and which should not," Pence wrote in his three-page letter addressed to members of Congress.
His dramatic break from Trump came minutes before the Republican vice president began presiding over a joint session of Congress, which is meeting to declare former Democratic Vice President Biden the victor.
Pence said in his letter that he shared "the concerns of millions of Americans about the integrity of this election," which Trump and his supporters have claimed without evidence was corrupted by widespread ballot fraud.
But Pence also wrote that "vesting the Vice President with unilateral authority to decide presidential contests would be entirely antithetical to" the system of check and balances between branches of the government designed by the framers of the Constitution.
"The Presidency belongs to the American people, and to them alone," Pence wrote.
"When disputes concerning a presidential election arise, under Federal law, it is the people's representatives who review the evidence and resolve disputes through a democratic process."
Trump less than two hours later blasted Pence for his action.
"Mike Pence didn't have the courage to do what should have been done to protect our Country and our Constitution, giving States a chance to certify a corrected set of facts, not the fraudulent or inaccurate ones which they were asked to previously certify," Trump wrote in a tweet.
"USA demands the truth!"
Trump's tweet came as protesters backing him stormed the Capitol and led to a suspension of debate, with Pence being ushered out of the Senate as a precaution.
As Pence released his statement, Trump was speaking at a rally outside of the White House, where he reiterated his call that the vice president undo Biden's election.
Trump and his allies claim that he and Pence lost to Biden and Vice President-elect Kamala Harris because of significant electoral ballot fraud in a handful of states that gave Biden his margin of victory.
"Mike Pence, I hope you're gonna stand up for the good of our Constitution and for the good of our country, and if you're not I'm gonna be very disappointed in you, I will tell you right now," Trump said at the rally.
"I'm not hearing good stories."
Multiple courts have rejected Trump's claims of fraud and election irregularities.
Shortly after Pence gaveled in the joint session of Congress, several Republicans objected to Arizona's Electoral College results in Biden's favor, triggering a period of debate.
GOP lawmakers are expected to object to the election results of several other states, but those efforts are also expected to merely delay the confirmation that Biden won the presidency, not block it.