Navratilova and singer Gavin DeGraw were both in jeopardy after ending up with the lowest scores after audience votes and points from the judges were combined.
Navratilova looked disappointed when she learned she was going home.
"It was an amazing experience," she said, after hugging partner Tony Dovolani. "I just wish it had lasted longer. I gave it everything I had but I blew it yesterday and it cost me."
She also wished the other competitors "great luck."
Navratilova's combined score from performances on Monday night and March 19 landed her on the bottom of the leader board. Welsh opera singer Katherine Jenkins was the top-scorer on both nights.
The tennis star missed some of the steps during her jive on Monday, and her foxtrot from the season debut, while thought to be elegant and refined, was "too careful," head judge Len Goodman told her.
Tonight's show also featured live performances by singer Matt Nathanson and the band Sugarland, as well as routines from the show's professional dancers.
Elimination in the competition results from a combination of judges' scores and audience votes. The celebrity with the lowest combined scores is sent home, until three stars remain. They and their partners compete for the championship.
Returning next week to perform are Disney star Roshon Fegan, "Extra" co-host Maria Menounos, NFL player Donald Driver, singer/actor Jack Wagner, DeGraw, actress Melissa Gilbert, Jenkins, R&B legend Gladys Knight, telenovela star William Levy, "The View" co-host Sherri Shepherd and "Family Matters" star Jaleel White.