In addition to regulating cell motility, contractility, and cytokinesis, the actin cytoskeleton plays a critical role in the regulation of transcription and gene expression. We have previously identified a novel muscle-specific actin-binding protein, STARS (striated muscle activator of Rho signaling), which directly binds actin and stimulates serum-response factor (SRF)-dependent transcription. To further dissect the STARS/SRF pathway, we performed a yeast two-hybrid screen of a skeletal muscle cDNA library using STARS as bait, and we identified two novel members of the ABLIM protein family, ABLIM-2 and -3, as STARS-interacting proteins. ABLIM-1, which is expressed in retina, brain, and muscle tissue, has been postulated to function as a tumor suppressor. ABLIM-2 and -3 display distinct tissue-specific expression patterns with the highest expression levels in muscle and neuronal tissue. Moreover, these novel ABLIM proteins strongly bind F-actin, are localized to actin stress fibers, and synergistically enhance STARS-dependent activation of SRF. Conversely, knockdown of endogenous ABLIM expression utilizing small interfering RNA significantly blunted SRF-dependent transcription in C2C12 skeletal muscle cells. These findings suggest that the members of the novel ABLIM protein family may serve as a scaffold for signaling modules of the actin cytoskeleton and thereby modulate transcription.