We induced gallstones in C57L mice fed with a high cholesterol diet and examined the expression of bile salt export pump (BSEP) on the canalicular membrane of hepatocytes and its relation with PKCα and HAX-1.Twenty-four gallstone-prone C57L mice were randomly assigned to receive a high cholesterol diet or a regular diet. Gallstone formation was recorded. BSEP, PKCα and phospho-PKCα expression was examined by immunoblotting assays. Co-expression of BSEP and HAX-1 was studied by immunofluorescent microscopy and immunoprecipitations. Gallstones were formed in all 12 mice fed with the high cholesterol diet. In Gallstone group, BSEP levels on the canalicular membrane of hepatocytes were markedly lower while a significant increase was observed in phosphorylated PKCα. Immunofluorescent microscopy showed that BSEP and HAX-1 were co-localized on the canalicular membrane, which was apparently enhanced by feeding with the high cholesterol diet. The immunoprecipitation assays further demonstrated that BSEP and HAX-1 showed enhanced interaction in the hepatocytes of mice fed with the high cholesterol diet. Cholesterol gallstone formation is associated with downregulation of BSEP expression on the canalicular membrane of hepatocytes with increased phosphorylation of PKCα. BSEP and HAX-1 show enhanced interaction with one another on the canalicular membrane during gallstone formation.