Chai-Qin-Cheng-Qi decoction (CQCQD) improves intestinal motility in acute pancreatitis (AP), but the mechanism(s) require elucidation. We investigated the effects of CQCQD and carbachol, a prokinetic agent, on colonic smooth muscle cells (SMCs) in L-arginine-induced necrotising AP model in rats. In treatment groups, intragastric CQCQD (20 g/kg, 2 hourly × 3 doses) or intraperitoneal carbachol (60 μg/kg) was given 24 hours after induction of AP. Both CQCQD and carbachol decreased the severity of pancreatic and colonic histopathology (all P < 0.05). Both CQCQD and carbachol reduced serum intestinal fatty acid binding protein, vasoactive intestinal peptide, and substance P and increased motility levels. CQCQD upregulated SMC phospholipase C-beta 1 (PLC-β1) mRNA and PLC protein (both P < 0.05), while both treatments upregulated protein kinase C-alpha (PKC-α) mRNA and PKC protein and downregulated adenylate cyclase (AC) mRNA and protein compared with no treatment (all P < 0.05). Neither treatment significantly altered L-arginine-induced PKC-β1 and PKC-ε mRNA reduction. Both treatments significantly increased fluorescence intensity of SMC intracellular calcium concentration [Ca2+]i (3563.5 and 3046.9 versus 1086.9, both P < 0.01). These data suggest CQCQD and carbachol improve intestinal motility in AP by increasing [Ca2+]i in colonic SMCs via upregulating PLC, PKC and downregulating AC.