BACKGROUND: Several Rho GTPase-activating proteins (RhoGAPs) are implicated in tumor progression through their effects on Rho GTPase activity. ARHGAP21 is a RhoGAP with increased expression in head and neck squamous cell carcinoma and with a possible role in glioblastoma tumor progression, yet little is known about the function of ARHGAP21 in cancer cells. Here we studied the role of ARHGAP21 in two prostate adenocarcinoma cell lines, LNCaP and PC3, which respectively represent initial and advanced stages of prostate carcinogenesis. RESULTS: ARHGAP21 is located in the nucleus and cytoplasm of both cell lines and its depletion resulted in decreased proliferation and increased migration of PC3 cells but not LNCaP cells. In PC3 cells, ARHGAP21 presented GAP activity for RhoA and RhoC and induced changes in cell morphology. Moreover, its silencing altered the expression of genes involved in cell proliferation and cytoskeleton organization, as well as the endothelin-1 canonical pathway. CONCLUSIONS: Our results reveal new functions and signaling pathways regulated by ARHGAP21, and indicate that it could contribute to prostate cancer progression.