Cyclin-dependent kinase inhibitor 3 (CDKN3) is proved to be associated with the progressing of many cancers. Whereas, its biological effects on prostate cancer (PC) are less understood. To investigate the functional mechanism of CDKN3 in PC, we examined the expression of CDKN3 in PC tissues and analyzed the disease free survival time of patients. We then transfected LNCaP and PC3 cells with siRNA-CDKN3 to silence CDKN3, and transfected 22RV1 and VCaP cells with full length CDKN3 cDNA for CDKN3 over-expression. Cell growth of these transfected cells were analyzed using CCK-8 assay. And transfected LNCaP and PC3 cells were further submitted to cell cycle, apoptosis, invasion and endogenous protein expression assays. We found that CDKN3 was highly expressed in PC and negatively correlated with disease relapse. And CDKN3 positively control the cell proliferation in prostate carcinoma cell lines. Knockdown of CDKN3 significantly promoted G1 phase arrest, elevated apoptosis rates, and suppressed cell invasion in both LNCaP and PC3 cells. Moreover, in vivo data showed that knockdown of CDKN3 expression dramatically inhibited the PC3 tumor growth in nude mouse model. Gene set enrichment analysis (GSEA) showed that cell cycle and DNA replication signaling were related with elevated CDKN3 expression. And results of western blot showed that the depletion of CDKN3 down-regulated the expression levels of cell cycle- and DNA replication-related proteins. In conclusion, our results highlight the importance of CDKN3 in PC and provide new insights into diagnostics and therapeutics of the PC.