Pseudomonas aeruginosa is an opportunistic pathogen that requires preexisiting epithelial injury to cause acute infections. We report that P. aeruginosa inhibits mammalian cytokinesis in a type III secretion system and exotoxin T (ExoT)-dependent manner. ExoT is a bifunctional type III secretion system effector protein that contains an N-terminal GTPase-activating protein domain and a C-terminal ADP-ribosyl transferase domain. Each of its domains inhibits cytokinesis in a kinetically, morphologically, and mechanistically distinct manner. The GTPase-activating protein-mediated inhibition of cytokinesis occurs early, likely as a consequence of its inhibitory effect on RhoA. The ADP-ribosyl transferase domain inhibits late steps of cytokinesis by blocking syntaxin-2 localization to the midbody, an event essential for completion of cytokinesis. These findings provide an example of a bacterial pathogen targeting cytokinesis.