The conservative radio show host's apology comes after lawmakers denounced his insults and several advertisers pulled out of his program.
Fluke, 30, testified on Capitol Hill, wanting her college health plan to cover contraceptives.
In a statement Limbaugh said, "My choice of words was not the best, and in the attempt to be humorous, I created a national stir. I sincerely apologize to Ms. Fluke for the insulting word choices."
Fluke has not commented.
Meantime, Republican presidential hopeful Newt Gingrich says Limbaugh was right to apologize for the comments.
Gingrich told CNN's "State of the Union" that he's glad Limbaugh issued the apology on Saturday and that it's time to move beyond the controversy. He said it's "silly" to suggest that Limbaugh speaks for the GOP, contending that members of the media are "trying desperately to protect" President Barack Obama.
The Associated Press contributed to this report.