The 37-year-old actress has previously worked with Whedon on his television shows Angel and Dollhouse, as well as his film adaptation of Much Ado About Nothing.
Acker will portray Audrey, a cellist and the love of lead character Phil Coulson's (Clark Gregg) life. The character was mentioned by name in an earlier episode of the show, but has not yet appeared in the series. Audrey believes Coulson is dead, and is unaware that the agent guards her from a distance.
The episode with Acker will air on Tuesday, April 22. Agents of S.H.I.E.L.D. returns to television at 8 p.m. ET on ABC Tuesday, April 1.