Cyclin-dependent kinase 5 (Cdk5) is known to regulate prostate cancer metastasis. Our previous results indicate that Cdk5 activates androgen receptor (AR) and supports prostate cancer growth. We also find that STAT3 is a target of Cdk5 in promoting thyroid cancer cell growth, while STAT3 may play a regulator to AR activation under cytokine control. This study is to investigate the regulation of Cdk5 and its activator p35 on STAT3/AR signaling in prostate cancer cells. Our results show that Cdk5 biochemically interacts with STAT3 and this interaction depends on Cdk5 activation in prostate cancer cells. The phosphorylation of STAT3 at serine-727 (p-S727-STAT3) is regulated by Cdk5 in cells and xenograft tumors. The mutant of STAT3 S727A reduces its interaction with Cdk5. We further show that the nuclear distribution of p-S727-STAT3 and the expression of STAT3-regulated genes (junB, c-fos, c-myc and survivin) are regulated by Cdk5 activation. STAT3 mutant does not further decrease cell proliferation upon Cdk5 inhibition, which implies that the role of STAT3 regulated by Cdk5 correlates to cell proliferation control. Interestingly, Cdk5 may regulate the interaction between STAT3 and AR through phosphorylation of S727-STAT3 and therefore up-regulate AR protein stability and transactivation. Correspondingly, clinical evidence shows that the level of p-S727-STAT3 is significantly correlated with Gleason score and the levels of upstream regulators (Cdk5 and p35) as well as downstream protein (AR). In conclusion, this study demonstrates that Cdk5 regulates STAT3 activation through S727 phosphorylation and further promotes AR activation by protein-protein interaction in prostate cancer cells.