Background Gambogic acid is a potent anticancer agent and has been found effective against various types of cancer cells. The present study was addressed to explore the cytotoxic potential of Gambogic acid and the modulation of autophagy and apoptosis in bladder cancer cells T24 and UMUC3. Methods Bladder cancer cell lines T24 and UMUC3 were treated with Gambogic acid, apoptosis was checked by flow-cytometry and expression of various autophagy and apoptosis related proteins was monitored by Western blotting. Confocal microscope was used for colocalization of p62 and Beclin-1. Results Gambogic acid induces reactive oxygen species, and elicits a strong autophagic response by activating JNK at earlier time points, which is inhibited at later time points with the activation of caspases. Reactive oxygen species mediated caspase activation causes degradation of autophagic proteins, cleavage of molecular chaperones (Hsp90 and GRP-78) and adaptor proteins (p62 and NBR1). Gambogic acid treatment results in mitochondrial hyperpolarization and cytochrome c release and activates caspases involved in both extrinsic and intrinsic apoptotic pathways. Gambogic acid abrogates NF-κB activation by ROS mediated inhibition of IκB-α phosphorylation. Functionally Gambogic acid induced autophagy acts as a strong cell survival response and delays caspase activation. Conclusion Our study provides the new insights about the mechanism of Gambogic acid induced modulation of autophagy and apoptosis in bladder cancer cells. All the molecular events responsible for Gambogic acid induced autophagy and apoptosis are mediated by reactive oxygen species. General significance Since Gambogic acid targets various cell survival molecules therefore, it may be considered as a potential anticancer agent against bladder cancer. [ABSTRACT FROM AUTHOR]