Objective To observe the effect of piperine (PA) on experimental gallstone formation in the gallbladder of C57BL/6 mice. Methods 3 dietary groups of C57BL/6 with 10 mice each group were allocated as control (normal mice chow),lithogenic (1% cholesterol diet) and PA (1% cholesterol diet + PA 30 mg/kg body weight) group respectively for 4 weeks. The expression of Scp2 gene in liver tissue was measured by RT-PCR and the bile lipid contents was measured chemically,the cholesterol saturation index (CSI) was calculated by Carey’s method. Results Cholesterol crystals and stones were found in 10/10 and 9/10 gallbladders respectively in lithogenic group,but not found in control and PA groups. Comparing with the lithogenic group,the expression of Scp2 gene in liver tissue and CSI in the gallbladder bile were significantly decreased in PA group. Conclusions PA inhibit the experimental gallstone formation induced by high cholesterol feeding in C57BL/6 mice,with simultaneous decreasing of both the Scp2 gene expression in the liver tissue and the CSI value in the gallbladder bile. The further study of the preventive effect of gallstong formation of PA,whether or not related to the above results,is strongly suggested.