The actress, who won an Oscar for her portrayal of June Carter Cash in Walk the Line in 2005, joined Chesney on stage at the House of Blues in New Orleans, where stars gathered to fund raise for Drew Brees and Matthew McConaughey's charities, the Dream Foundation and the Just Keep Livin' Foundation.
Chesney, 46, brought Witherspoon on stage to help him perform an acoustic version of Johnny Cash's "Jackson," and the crowd loved her Southern twang as she hit the song's final note.
We know Witherspoon loves the Man in Black – besides playing his wife on screen, she's photographed her 20-month-old son Tennessee "crawling the line" – and it seems she's a fan of Chesney, too.
"Singing with Kenny C #NOLA #amazingnight #jkl," Witherspoon, 38, captioned an Instagram of their musical moment.
Watch Witherspoon take the mic below.