Silibinin, a natural flavonolignan, induces apoptosis in human bladder transitional-cell papilloma RT4 cells both in vitro and in vivo; however, mechanisms of such efficacy are not completely identified. Here, we studied the mechanisms involved in silibinin-induced apoptosis of RT4 cells having intact p53. Silibinin increased p53 protein level together with its increased phosphorylation at serine 15, activated caspase cascade and caused Bid cleavage for apoptosis. Silibinin-caused p53 activation was mediated via ATM-Chk2 pathway, which in turn induced caspase 2-mediated apoptosis. Pifithrin-alpha, a p53 inhibitor, reversed silibinin-induced caspase activation including caspase 2; however, caspase 2 inhibitor also reversed p53 phosphorylation suggesting a bidirectional regulation between them. Further, silibinin caused a rapid translocation of p53 and Bid into mitochondria leading to increased permeabilization of mitochondrial membrane and cytochrome c release into the cytosol. JNK1/2 activation was observed as a connecting link for p53-mediated caspase 2 activation. Interestingly, silibinin-induced apoptosis was mediated, in part, via Cip1/p21 cleavage by caspase, which was reversed by Cip1/p21 siRNA. Together, these results suggested the novel mechanisms for apoptosis induction by silibinin involving p53-caspase 2 activation and caspase-mediated cleavage of Cip1/p21.