The prostate cancer is one of the leading causes of men's cancer mortality. The development of alternative chemotherapeutic strategies is urgent. Berberine has displayed significant anti-prostate cancer activities. The underlying mechanisms are not fully understood. In the current study, we found that berberine induced apoptosis and programmed necrosis in cultured prostate cancer cells (LNCaP and PC-82 lines), and necrosis weighted more than apoptosis in contributing berberine's cytotoxicity. We demonstrated that mitochondrial protein cyclophilin-D (Cyp-D) is required for berberine-induced programmed necrosis. Inhibition of Cyp-D by its inhibitors cyclosporin A (CSA) or sanglifehrin A (SFA), and by Cyp-D shRNA depletion alleviated berberine-induced prostate cancer cell necrosis (but not apoptosis). Our data found that in prostate cancer cells, berberine induced reactive oxygen species (ROS) production, which dictated P53 translocation to mitochondria, where it physically interacted with Cyp-D to open mitochondrial permeability transition pore (mPTP). The anti-oxidant N-acetylcysteine (NAC), the P53 inhibitor pifithrin-α (PFTα) as well as P53 siRNA knockdown suppressed berberine-induced P53 mitochondrial translocation and Cyp-D association, thus inhibiting mitochondrial membrane potential (MMP) decrease and prostate cancer cell necrosis. In summary, the results of the present study provide mechanistic evidence that both apoptosis and programmed necrosis attribute to berberine's cytotoxicity in prostate cancer cells.