Advanced and hormone-refractory prostate cancer has long been considered as a chemoresistant disease. Recently, it was found that 14-3-3sigma expression increases as prostate tumor progresses, and that 14-3-3sigma contributes significantly to drug resistance in breast cancers. We, thus, hypothesized that advanced and hormone-refractory prostate cancers may have an increased level of 14-3-3sigma, which in turn may contribute to drug resistance in advanced and hormone-refractory prostate cancers. In this study, we tested this hypothesis and found that, indeed, the expression level of 14-3-3sigma in androgen-independent prostate cancer cell lines DU145, PC3, and CWR22RV are much higher than that in the androgen-dependent cell line LNCaP, and that the androgen-independent cells are more resistant to mitoxantrone and Adriamycin than the androgen-dependent cells. Depleting 14-3-3sigma expression in DU145 and CWR22RV by RNA interference significantly sensitized these cells to mitoxantrone and Adriamycin by abrogating G2-M checkpoint and increasing apoptosis, whereas restoring 14-3-3sigma expression in LNCaP cells enhanced drug resistance. We also showed that 14-3-3sigma deficiency caused nuclear localization of Cdc2 and dephosphorylation of the Tyr15 residue upon DNA damage. Based on these studies, we propose that therapeutic intervention targeting 14-3-3sigma may be useful for sensitizing hormone-refractory prostate cancers to chemotherapy by both G2-M checkpoint abrogation and apoptosis enhancement.