Protein kinase D (PKD) is a family of novel diacylglycerol/phorbol ester targets that regulate many important cellular functions including cell growth and survival. We now provide experimental evidence to indicate that PKD3 contributes to prostate cancer cell growth and survival. Expression of PKD3 as well as PKD1 was significantly higher in human prostate tumors compared with normal tissues as revealed by immunohistochemistry. Moreover, PKD3 exhibited a marked increase in nuclear localization in tumor tissues, which correlated with tumor grade. Isoforms of PKD were differentially expressed and localized between normal and human prostate cancer cells. Increased protein expression and nuclear accumulation of PKD3 were observed in the more aggressive androgen-independent PC3 and DU145 cells compared with the less aggressive androgen-dependent LNCaP cells. Overexpression of wild-type PKD3 in LNCaP cells blocked phorbol 12-myristate 13-acetate (PMA)-induced apoptosis in association with inhibition of PMA-induced down-regulation of Akt activity, and prolonged extracellular signal-regulated kinase (ERK)1/2 activation. Overexpression of wild-type PKD3 also promoted S phase entry, whereas depletion of endogenous PKD3 resulted in G(0)-G(1) phase cell cycle arrest and inhibition of PC3 cell proliferation. In PC3 and DU145 cells, PKCepsilon regulated PKD3 kinase activity and nuclear localization. Moreover, ectopical expression of PKD3 increased, whereas depletion of endogenous PKD3 reduced basal Akt and ERK1/2 activities. Further analysis showed that up-regulation of Akt activity induced by PKD3 required phosphatidylinositol-3-OH kinase and p38. In summary, our data indicate that PKD3 contributes to growth and survival of prostate cancer cells and may represent a novel therapeutic target for prostate cancer.