Release of proinflammatory cytokines or other mediators from immune cells or intestinal epithelial cells is a critical activation event in C. difficile toxin‐mediated intestinal inflammation. In this study we investigated the role of TNF‐α in the pathogenesis of CDI using mouse model of toxin exposure or CDI, and dissected the roles of RhoGTPase and MAPK in TcdA‐induced production of TNF‐α by macrophages. We showed that inactivation of RhoA by TcdA activates p38 and ERK, which controls of TNF‐α production by macrophage Raw264.7 cells. We also demonstrated that calcium signaling regulates TcdA‐induced TNF‐α in macrophage cell line. Based on these findings we propose that inhibition of p38 and ERK activation may be beneficial in reducing inflammatory symptoms in patients with CDI by blocking toxin‐induced TNF‐α release.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.