Vibrio parahaemolyticus type III secretion system 2 (T3SS2) is essential for the organism’s virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. Here, we identify a type III secretion system (T3SS2)-secreted effector, VopZ, that is essential for V. parahaemolyticus pathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticus colonization, whereas deletion of VopZ amino acids 38–62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase TAK1 and thereby prevents the activation of MAPK and NF-κB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1. Collectively, our findings suggest that VopZ’s inhibition of TAK1 is critical for V. parahaemolyticus to induce diarrhea and intestinal pathology. Display omitted •VopZ is identified as a multifunctional V. parahaemolyticus T3SS2 effector•V. parahaemolyticus colonization can be uncoupled from diarrhea and tissue damage•VopZ inhibits TAK1 activation and thereby prevents NF-κB and MAPK activation•VopZ’s inhibition of TAK1 is essential for intestinal pathology, but not colonization Waldor and colleagues have identified a multifunctional type III secretion system 2 (T3SS2)-secreted effector, VopZ, with distinct and essential roles in V. parahaemolyticus intestinal colonization and diarrheagenesis. VopZ inhibits activation of the kinase TAK1 and thereby prevents activation of MAPK and NF-κB signaling pathways, which lie downstream. VopZ’s activity against TAK1 signaling appears to be essential for V. parahaemolyticus-induced diarrhea and intestinal pathology; however, genetic analyses indicate that a distinct VopZ activity is essential for intestinal colonization.