Technical Abstract: The current study investigated the mechanism of immune regulation of IL-25 and the contribution of IL-25 to nematode infection-induced alterations in intestinal smooth muscle and epithelial cell function. Mice were infected with an enteric nematode or injected with IL-25 or IL-13. In vitro smooth muscle contractility was performed to assess muscle responses to acetylcholine, serotonin, and electrical field stimulation, as well as spontaneous contractions. Mucosal function was determined by challenging the muscle-free mucosa mounted in Ussing chambers for epithelial secretion, or in micro-snap well for trans-epithelial resistance. Gene expression of major cytokines were determined via real-time PCR or viewed by immune fluorescent staining. Epithelial cells, but not immune cells, constitute the major source of IL-25 in the small intestine constitutively and during nematode infection. Infection-induced up-regulation of IL-25 expression depended upon IL-13 activation of Stat6. IL-25 deficiency in mice caused an impaired Th2 immunity against N. brasiliensis infection, associated with an abolished intestinal smooth muscle hyper-contractility and reduced mucosal functional response, whereas exogenous IL-25 induced characteristic changes in intestinal function similar to that induced by nematode infection. Exogenous IL-25 was unable to restore the impaired host immunity against N. brasiliensis infection in IL-13-/- mice. Thus, epithelial-derived IL-25 plays a critical role in nematode infection-induced changes in intestinal function and IL-13 is the major Th2 cytokine responsible for IL-25-mediated changes in gut function.