miR-331-3p blocks AR signaling via inhibition of ERBB-2 expression and AKT activity in prostate cancer cells. AR antagonists such as bicalutamide bind to the AR and prevent its activation and expression of AR target genes, such as PSA. Nevertheless, AR signaling may persist in prostate cancer cells despite AR blockade, in part via increased expression of the ERBB-2 receptor tyrosine kinase and subsequent activation of the PI3K/AKT pathway, which causes AR phosphorylation and promotes expression of AR target genes. miR-331-3p directly targets the ERBB-2 mRNA 3′-UTR to regulate ERBB-2 protein expression, thereby reducing PI3K/AKT signaling and AR signaling. The combination of an AR antagonist (bicalutamide) and miR-331-3p effectively blocks AR signaling (PSA expression and PSA promoter activity) in LNCaP prostate cancer cells. (+) indicates activation step of pathway and (−) indicates inhibition of pathway component.