The androgen receptor (AR), a nuclear transcription factor, mediates male sexual differentiation, and its excessive action is associated with prostate cancer. We have characterized a negative regulatory domain in the AR hinge region, which interacted with filamin A (FLNa), an actin-binding cytoskeletal protein. FLNa interfered with AR interdomain interactions and competed with the coactivator transcriptional intermediary factor 2 to specifically down-regulate AR function. Although full-length FLNa was predominantly cytoplasmic, a C-terminal 100-kDa fragment of FLNa colocalized with AR to the nucleus. This naturally occurring FLNa fragment repressed AR transactivation and disrupted AR interdomain interactions and transcriptional intermediary factor 2-activated AR function in a manner reminiscent of full-length FLNa, raising the possibility that the inhibitory effects of cytoplasmic FLNa may be transduced through this fragment, which can localize to the nucleus and form part of the pre-initiation complex. This unanticipated role of FLNa adds to the growing evidence for the involvement of cytoskeletal proteins in transcription regulation.