"It is with great humility that I do so, knowing the tremendous honor it has been to serve" in the leadership of the House of Representatives, said Cantor, who spoke to reporters after formally announcing his resignation to House Republicans in a closed-door meeting.
Urging Republicans to "put minor differences aside" and elect a GOP House, he promised to continue to be "a champion for conservatives across the nation who are dedicated to preserving liberty."
"I may have suffered a personal setback last night, I couldn't be more optimistic about the future of this country," he added.
His resignation from the post is effective July 31, but Republicans will hold the election to replace him on June 19, an aide said.
Asked if his defeat means a death knell for immigration reform, Cantor reiterated that he believes there is common ground to fix a broken system.
The decision sent shockwaves through Capitol Hill as members of the conservative and establishment wings of the Republican Party jockey to fill the influential post.
Cantor had previously been discussed as a potential successor to House Sepaker John Boehner if the current speaker stepped down.
Now, other top Republicans are already floating names to replace Cantor in the high-ranking job.
Majority Whip Rep. Kevin McCarthy is widely viewed as a likely candidate for the job, and Cantor said Wednesday that McCarthy will get his backing if he decides to pursue the position of majority leader.
Rep. Pete Sessions, R-Texas, is actively discussing his interest in the post too.
Rep. Jeb Hensarling of Texas, former GOP Conference Chairman and current Chairman of the House Financial Services Committee, is another favorite of conservatives for the job. In a statement Monday, Hensarling acknowledged that he's mulling a run.
"I am humbled by the many people who have approached me about serving our Republican Conference in a different capacity in the future. There are many ways to advance the causes of freedom and free enterprise, and I am prayerfully considering the best way I can serve in those efforts," he said.
Budget chief Paul Ryan, known outside the Beltway as Mitt Romney's running mate during the 2012 election, reiterated to reporters Wednesday that he is not interested in the post.
—By Luke Russert and Frank Thorp V, NBC News. NBC's Carrie Dann contributed.