Clostridium difficile (C. difficile) is an opportunistic bacteria that flourishes in intestinal flora is altered by antibiotic use and can cause large intestinal colitis if left untreated. The mechanism of MAVS-mediated innate immune signaling in response to C. difficile infection remains unclear. This knowledge gap was addressed in the present research by administration of an antibiotic cocktail to WT and MAVS-deficient (MAVS-/-) mice for five days, followed by the oral administration of C. difficile VPI 10,463 strain (1 x 10(7) colony forming units). Subjects were subsequently observed for another eight days for signs of colitis. Colon and cecum tissue samples were harvested from naive and infected mice two days post-infection for histopathologic analysis. Colon tissue samples were harvested to analyze cytokine gene expression and RegIII beta/gamma gene expression. MAVS-deficient mice suffered significantly higher mortality rates as well as increased mucosal tissue inflammation and damage after C. difficile infection (P < 0.05). MAVS-/- mice displayed a significantly greater increase in IL-6, IL-1 beta, TNF-alpha and IFN-beta expression in colon tissues in contrast to WT mice challenged with C. difficile (P < 0.05). RegIII beta/gamma gene expression was severely attenuated in the colons of MAVS-/- mice (P < 0.05). These findings underscore MAVS as a vital innate immune system mediator in the intestinal mucosa and further suggests that MAVS-mediated maintenance of the intestinal epithelial barrier is an important defense against enteric pathogens.