Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming bacterial toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and uses a MARTX toxin with three effector domains-an actin cross-linking domain (ACD), a Rho inactivation domain (RID), and an / hydrolase domain (ABH)-to suppress innate immunity and enhance colonization. We investigated whether these multiple catalytic enzymes delivered from a single toxin functioned in a coordinated manner to suppress intestinal innate immunity. Using cultured human IECs, we demonstrated that ACD-induced cytoskeletal collapse activated extracellular signal-regulated kinase, p38, and c-Jun amino-terminal kinase mitogen-activated protein kinase (MAPK) signaling to elicit a robust proinflammatory response characterized by the secretion of interleukin-8 (IL-8; also called CXCL8) and the expression of CXCL8, tumor necrosis factor (TNF), and other proinflammatory genes. However, RID and ABH, which are naturally delivered together with ACD, blocked MAPK activation through Rac1 and thus prevented ACD-induced inflammation. RID also abolished IL-8 secretion induced by heat-killed bacteria, TNF, or latrunculin A. Thus, MARTX toxins use enzymatic multifunctionality to silence the host response to bacterial factors and to the damage caused by the toxins. Furthermore, these data show how V. cholerae MARTX toxin suppresses intestinal inflammation and contributes to cholera being classically defined as a noninflammatory diarrheal disease. Copyright 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.