Silibinin is known for its hepatoprotective, anti-inflammatory, and anti-carcinogenic effects. We found that silibinin exhibited a protective effect against chemotherapeutic reagent mitomycin C-induced cell death in A375-S2 cells in a p53-dependent manner, which contradicted the findings of previous studies investigating the anti-neoplastic activity of silibinin and developing silibinin as a potential anti-neoplastic drug in clinical therapy. Mitomycin C administration triggered a time- and dose-dependent cell death in A375-S2 cells. Apoptotic morphology, DNA fragmentation, and caspase-3 activation demonstrated that the major cause of A375-S2 cell death by mitomycin C was apoptosis. This was associated with a marked increase of p53 level and changes in mitochondria associated proteins. However, preincubation with silibinin prior to mitomycin C treatment substantially suppressed cell apoptosis, attenuated the change of p53 and Bcl-2 expressions, blocked the translocation of Bax to mitochondrial outer membrane, and ameliorated the loss of mitochondrial membrane potential, but mitomycin C stimuli led to few changes in the protein levels of caspase 8, Fas ligand, and Fas-associated death domain protein, indicating that silibinin protected cells from mitomycin C-induced apoptosis mainly via suppressing the mitochondria-mediated intrinsic apoptosis pathway, but not in an extrinsic manner.