Hsp90 is widely overexpressed in cancer cells and believed to be essential for the maintenance of malignant phenotypes. Targeting Hsp90 by small molecules has shown promise in solid and hematologic malignancies, which likely involves degradation of client oncoproteins in a cell-type-specific manner. In this study, we found that structurally unrelated Hsp90 inhibitors induce DNA damage and apoptosis via p53-dependent induction of PUMA, which indirectly triggers Bax activation and mitochondrial dysfunction in colon cancer cells. Deficiency in PUMA, BAX, or p53, at lesser extent, abrogated 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced apoptosis and mitochondrial dysfunction, and enhanced clonogenic cell survival. Furthermore, suppression of p53-dependent p21 induction or enhanced p53 activation synergized with 17-AAG to induce PUMA-dependent apoptosis. Finally, PUMA was found to mediate apoptotic and therapeutic responses to the 17-AAG analog 17-DMAG in xenografts. These results show an important role of the p53/PUMA/Bax axis in Hsp90 inhibitor-induced killing of p53 wild-type cells, and have important implications for their clinical applications.