The curcumin analogue B5 is a thioredoxin reductase (TrxR) inhibitor, which has been shown to exhibit more potential anti-cancer activity than curcumin. However, the molecular mechanisms are needed to further elucidate. In this study, we demonstrate that B5 has inhibitory activities against two human cervical cancer cell lines, CaSki and SiHa, by induction of apoptosis, indicated by the down-regulation of XIAP, activation of caspase-3, caspase-8 and caspase-9 cascade and the subsequent cleavage of PARP. B5-induced apoptosis is mediated by the mitochondrial pathway, indicated by the destruction of mitochondrial membrane potential (MMP), down-regulation of Bcl-xL and up-regulation of Bid and Bim. Trx system is found to play an important role in B5-induced apoptosis in CaSki and SiHa cells. The treatment of B5 results in the shift of thioredoxin (Trx) redox state to the oxidized form and subsequent ROS accumulation. The addition of antioxidant N-acetylcysteine significantly blocks B5-induced apoptosis. Furthermore, Oxidized Trx accumulation leads to the activation of apoptosis signal-regulating kinase 1 (ASK1), followed by the activation of downstream p38/JNK mitogen-activated protein kinases (MAPK). On the other hand, B5 is observed to induce autophagy in CaSki and SiHa cells via the inhibition of PI3K/Akt/mTOR/4EBP1 signaling pathway. B5-induced autophagy has pro-apoptotic effect in SiHa cells as evidenced by the addition of autophagy inhibitor. Token together, these data provide a possible molecular mechanism for the anti-cancer effect of B5 on CaSki and SiHa cells and suggest potential application of B5 as anti-cancer agent for human cervical cancer.