Abstract: Objective To investigate the changes of gastrointestinal migrating myoelectric complex (MMC) and bile acid pool size during cholesterol gallstone formation in guinea pigs. Methods Seventy-five guinea pigs were randomized into 5 equal groups and given normal diet for 8 weeks, 1% cholesterol diet for 2, 4, and 8 weeks, and cholesterol diet with Vitamin C for 8 weeks, respectively. At the end of the feeding period, the gallbladder motility was assessed by deploying 4 bipolar silver electrodes on the antrum, duodenum, jejunum and ileum. Seven days later, MMC was recorded and the total bile acid pool size measured according to the isotope dilution method. Results After cholesterol feeding, the gallbladder motility declined in 4- and 8-week groups, which showed a reduction of bile acid pool size by 42.75% (P<0.05) and 57.24% (P<0.05), respectively. Compared to the control group, MMCs started mostly in the duodenum. MMC cycle duration was prolonged in the duodenum by 1.32 times in 4-week group (P<0.05) and 1.44 times in 8 week group (P<0.05) whereas MMC amplitude was lowered in the duodenum by 40.91% in 4-week group (P<0.05) and 60.17% in 8 week group (P<0.05). Supplementation of vitamin C caused changes in MMC similar to those observed in the guinea pigs of 2-week group. Conclusions High-cholesterol diet inhibits MMC activity and reduces bile acid pool size in guinea pigs, which, along with impairment of the gallbladder motility, contributes to the formation of cholesterol stone, whereas vitamin C supplement in the diet may help prevent cholelithiasis.