"James, I just know that you have this performance bug about you that we've got to expose to the world," said Seacrest before asking the actor, 41, to step to the mic.
"He's stage-starved," called out Lady A's Charles Kelley. "I love it!"
"I'm really nervous," said Marsden, a big Brooks fan, just before he started crooning. But he won over the room as soon as he sang the first few notes.
"Now I'm just pissed because he's better than me," joked Kelley, 33.
The country trio – and even Seacrest himself – joined in on the fun and helped Marsden, who sang on screen in 2007's Hairspray, with the chorus. "It's an all-sing," Hillary Scott declared.
Marsden's The Best of Me, based on the Nicholas Sparks book, is in theaters Oct. 17. The film's soundtrack, which includes Lady A, is available now.