Star Milla Jovovich announced the actress's return to the series on Twitter.
Jovovich tweeted: "So to answer many of your questions in one fell swoop, yes! The gorgeous and talented Sienna Guillory is back as Jill Valentine in RE4!!!"
Guillory played the role in 2004's Resident Evil: Apocalypse.
Resident Evil: Afterlife, directed by Paul W.S. Anderson, also stars Ali Larter, Wentworth Miller and Shawn Roberts.
The 3D picture opens September 10 in US cinemas.