Pro-inflammatory cytokine, IL-1, contributes to the reduced contractile responses of gut smooth muscle observed in both animal colitis models and human inflammatory bowel disease. However, the mechanisms are not well understood. The effects of IL-1 on the signaling targets mediating acetylcholine(ACh)-induced initial and sustained contraction were examined using rabbit colonic circular muscle strips and cultured muscle cells. The contraction was assessed through cell length decrease, MLC₂₀ phosphorylation and the activation of PLC- and Rho kinase. Expression levels of the signaling targets were determined by Western blot and real-time RT-PCR. The small interfering RNAs (siRNAs) for Regulator of G protein Signaling 4 (RGS4) were used to silence endogenous RGS4 in muscle strips or cultured muscle cells. IL-1 treatment of muscle strips inhibited both initial and sustained contraction and MLC₂₀ phosphorylation in isolated muscle cells. IL-1 treatment increased RGS4 expression, but had no effect on muscarinic receptor binding or G_q expression. In contrast, IL-1 decreased the expression and phosphorylation of CPI-17, but had no effect on RhoA expression or ACh-induced Rho kinase activity. Up-regulation of RGS4 and down-regulation of CPI-17 by IL-1 in the muscle strips were corroborated in cultured muscle cells. Knockdown of RGS4 by siRNA in both muscle strips and cultured muscle cells blocked the inhibitory effect of IL-1 on initial contraction and PLC- activation, whereas overexpression of RGS4 inhibited PLC- activation. These data suggest that IL-1 up-regulates RGS4 expression resulting in the inhibition of initial contraction and down-regulates CPI-17 expression during the sustained contraction in colonic smooth muscle.