Objective To discuss the relationship between motilin, vasoactive intestinal peptide and the gallstone formation. Methods The level of motilin, vasoactive intestinal peptide in plasma, bile and gallbladder tissue of 48 cases of chololithiasis before operation and the first, third, seventh day after cholecystectomy were mesured by radioimmunoassay. Results The level of motilin in plasma was markedly increased in patients with chololithiasis before cholecystectomy and the first day after cholecystectomy. The level of motilin, vasoactive intestinal peptide in bile and gallbladder tissue were significantly increased in patients and motilin was positively correlated with vasoactive intestinal peptide in the gallbladder tissue. Conclusion Motilin, vasoactive intestinal peptide might affect the gallstone formation by affecting the motility of gallbladder.