Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immunocompromised individuals and cystic fibrosis patients. ExoS and ExoT are two homologous bifunctional Type III Secretion System (T3SS) virulence factors that induce apoptosis in target host cells. They possess a GTPase Activating Protein (GAP) domain at their N-termini, which share ~76% homology, and an ADP-ribosyltransferase (ADPRT) domain at their C-termini, which target non-overlapping substrates. Both the GAP and the ADPRT domains contribute to ExoTs cytotoxicity in target epithelial cells, whereas, ExoS-induced apoptosis is reported to be primarily due to its ADPRT domain. In this report, we demonstrate that ExoS/GAP domain is both necessary and sufficient to induce mitochondrial apoptosis. Our data demonstrate that intoxication with ExoS/GAP domain leads to enrichment of Bax and Bim into the mitochondrial outer-membrane, disruption of mitochondrial membrane and release of and cytochrome c into the cytosol, which activates initiator caspase-9 and effector caspase-3, that executes cellular death. We posit that the contribution of the GAP domain in ExoS-induced apoptosis was overlooked in prior studies due to its slower kinetics of cytotoxicity as compared to ADPRT. Our data clarify the field and reveal a novel virulence function for ExoS/GAP as an inducer of apoptosis.