YopE, a type III secretion effector of Yersinia pseudotuberculosis , is essential for virulence. YopE inactivates Rho GTPase with its GTPase activating protein (GAP) activity. Since Rho GTPases regulate immune functions, such as phagocytosis, reactive oxygen species (ROS) production, and cytokine production, the impact of YopE on Yersinia colonization could be due to a number of events. Different bacterial GAPs are structurally homologous but inactivate different subsets of targets. What confers their specificities was investigated in this work. The YopE-Rac1 structure model was used to predict YopE residues interfacing Rac1. YopE mutants were constructed and tested in cell culture assays specific for different Rho GTPases, Rac1-dependent phagocytosis inhibition, RhoA-dependent cell rounding, and Rac2-dependent ROS inhibition. Mutation at residues F102, I106, L109, and F156 resulted in differential GAP activities. These results combined with structural modeling suggest that these positions distinguish among different Rho GTPases by interacting with conserved residues in the switch regions that adopt slightly different conformations in different Rho GTPases. To identify which GAP activities are critical for Yersinia colonization in different tissues, we infected mice with the L109A mutant, which retains WT level antiphagocytosis activity and intermediate ROS inhibition and cell rounding activity, and ESptP mutant, which retains high antiphagocytosis activity but no ROS inhibition or cell rounding activity. We determined that WT levels of antiphagocytic activity and ROS inhibition activity, not cell rounding activity, are needed for spleen colonization while full levels of ROS inhibition activity and cell rounding activity are not required for colonization of intestinal tissues and lymph nodes. In addition, we found that GAP activity is not the only way YopE confers virulence. A strain expressing catalytically inactive YopER144A was more virulent than ΔyopE and another inactive mutant, YopEG152E. Genetic data and structural modeling suggest that long side chain of glutamic acid at position 152 might disrupt binding with Rho GTPases. Interestingly, yopER144A can cause mild cell rounding at high MOI. yopER144A might achieve its virulence by competitively inhibiting Rho GTPases. In summary, critical functions of YopE during infection include inhibition of ROS in spleen and inhibition of phagocytosis in intestinal tissues, and a mutant lacking GAP activity can still cause virulence potentially by competitively inhibiting Rho GTPases.