The intratumoral synthesis of androgen from cholesterol or the conversion of adrenal precursor androgens to active androgens represent two important mechanisms underlying the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC).Aldo-ketoreductase family 1 member C3 (AKR1C3), as a key steroidogenicenzyme, is overexpressed in PCa and is associated with the development of CRPC, and moreover, in our study AKR1C3 exhibited a positive correlation with the Gleason Score which further strengthen the potential biomarker role of AKR1C3 for PCa progression.In our previous study, berberine (BBR) could delay the latent periods to the progression of CRPC in castrated nu/nu mice bearing a subcutaneous LNCaPxenograft.However, the mechanisms of BBR on CRPC is still under investigation.In this study, we found BBR could inhibit 22RV1 prostate cancer cell growth, decrease the formation of total testosterone (T), suppress AKR1C3 enzyme activity without influence its mRNA or protein expression.Virtual screening demonstrated that BBR could enter the active center of AKRIC3 and form Pi-Pi interaction with the amino-acid residue Phe306 and Phe311.This structural interaction attributed to the suppression of AKR1C3 enzyme activity and inhibition of 22RV1 prostate cancer cell growth by decreasing the intracellular androgen synthesis.Our study reassure that AKR1C3 may be a potential anti-cancer drug targe for CRPC and the clarification of mechanisms of BBR on CRPC would provide the experimental basis for the design, research and development of AKR1C3 inhibitors using BBR as the lead compound.