Motility disorders are frequently observed in intestinal inflammation. We previously reported that in vitro treatment of intestinal smooth muscle tissue with IL-1 decreases the expression of CPI-17, an endogenous inhibitory protein of smooth muscle serine/threonine protein phosphatase, thereby inhibiting contraction. The present study was performed to examine the pathophysiological importance of CPI-17 expression in the motility disorders using an in vivo model of intestinal inflammation and to define the regulatory mechanism of CPI-17 expression by pro-inflammatory cytokines. After induction of acute ileitis with TNBS, CPI-17 expression declined in a time-dependent manner. Parallel with this decrease in CPI-17, cholinergic agonist-induced contraction of strips of smooth muscle and sensitivity of permeabilized smooth muscle fibers to Ca²⁺ were reduced. Among the various pro-inflammatory cytokines tested, TNF- and IL-1 were observed to directly inhibit CPI-17 expression and contraction, in cultured rat intestinal tissue. Moreover, both TNF- and IL-1 inhibited CPI-17 expression and contraction of smooth muscle tissue isolated from wild-type and IL-1,double KO mice. However, IL-1-treatment failed to inhibit CPI-17 expression and contraction in TNF- KO mice. These findings suggest that CPI-17 was down-regulated during intestinal inflammation, and that TNF- plays a central role in this process. Down-regulation of CPI-17 may play a role in motility impairments in inflammation.