< i>Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury. In cultured cells, toxin A inactivates Rho proteins by monoglucosylation. In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms. We investigated the role of mitogen-activated protein (MAP) kinases in these events. In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascades within 1 to 2 minutes. Activation of p38 was sustained, whereas stimulation of extracellular signal-regulated kinases and c-Jun NH₂-terminal kinase was transient. Rho glucosylation became evident after 15 minutes. IL-8 gene expression was reduced by 70% by the MEK inhibitor PD98059 and abrogated by the p38 inhibitor SB203580 or by overexpression of dominant-negative mutants of the p38-activating kinases MKK3 and MKK6. SB203580 also blocked monocyte necrosis and IL-1β release caused by toxin A but not by other toxins. Finally, in mouse ileum, SB203580 prevented toxin A–induced neutrophil recruitment by 92% and villous destruction by 90%. Thus, in monocytes exposed to toxin A, MAP kinase activation appears to precede Rho glucosylation and is required for IL-8 transcription and cell necrosis. p38 MAP kinase also mediates intestinal inflammation and mucosal damage induced by toxin A.