Initial Ca²⁺-dependent contraction of intestinal smooth muscle is inhibited upon IL-1 treatment. The decrease in contraction reflects up-regulation of RGS4 via a canonical IKK2/IB/NF-B pathway. Here we show that activation of various protein kinases, including extracellular signal-regulated kinases (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) differentially modulates IL-1-induced up-regulation of RGS4 in rabbit colonic muscle cells. IL-1 treatment caused a transient phosphorylation of ERK1/2 and p38 MAPK. It also caused phosphorylation of Akt and GSK3, sequential downstream effectors of PI3K. Pretreatment with PD98059 (an ERK inhibitor) and SB203580 (a p38 MAPK inhibitor) significantly inhibited IL-1-induced RGS4 expression. In contrast, LY294002 (a PI3K inhibitor) augmented whereas GSK3 inhibitors inhibited IL-1-induced RGS4 expression. PD98059 blocked IL-1-induced phosphorylation of IKK2, degradation of IB, and phosphorylation and nuclear translocation of NF-B subunit p65, whereas SB203580 had a marginal effect, implying that effect of ERK1/2 is exerted on the canonical IKK2/IB/p65 pathway of NF-B activation but the effect of p38 MAPK may not predominantly involve the NF-B signaling. The increase in RGS4 expression enhanced by LY294002 was accompanied by increase in phosphorylation of IKK2/IB/p65, and blocked by pretreatment with the inhibitors of IKK2 (IKK2-IV) and IB (MG132). Inhibition of GSK3 abolished IL-1-induced phosphorylation of IKK2/p65. These findings suggest that ERK1/2 and p38 MAPK enhance IL-1-induced upregulation of RGS4; the effect of ERK1/2 reflects its ability to promote IKK2 phosphorylation and increase NF-B activity. GSK3 acts normally to augment activation of the canonical NF-B signaling. The PI3K/Akt/GSK3 pathway attenuates IL-1-induced up-regulation of RGS4 expression by inhibiting NF-B activation.