Indirubin-3'-monoxime (I3M) is a derivative of indirubin, an active component from a Chinese medicinal recipe with known anti-cancer function. I3M has been well established as a cyclin-dependent kinase (CDK) inhibitor, while the molecular mechanism underlying I3M-induced apoptosis has not been fully elucidated. In this study, we focused on the critical role of the pro-apoptosis Bcl-2 family members in I3M-induced apoptosis. We first observed I3M-induced apoptosis in a time- and dose-dependent manner in three different types of human cancer cells-cervical cancer HeLa, hepatoma HepG2 and colon cancer HCT116. Induction of the caspase cascade for both the extrinsic and intrinsic pathways was demonstrated, including caspase-8, -9 and -3 activation. Initiation of the death receptor pathway started with enhanced surface expression of DR4 and DR5, as well as increased total protein level, which correlated with the up-regulation of p53 and its transcriptional activity. Importantly, we found in HeLa cells that caspase-8 activation resulted in Bid cleavage, followed by Bax conformational change and hence the amplification of the apoptotic signals through the mitochondrial pathway. Consistently, stable knockdown of Bid abrogated I3M-induced Bax conformational change and cell death. Moreover, ectopic expression of a viral caspase inhibitor (CrmA) or Bcl-2 partially protected I3M-induced apoptosis. In conclusion, our results indicate that I3M mainly elicites apoptosis through extrinsic pathway with type II response mediated by the pro-apoptotic Bcl-2 family members (Bid and Bax).