OBJECTIVE To regain bile excretory function of hepatocytes cultured in vitro. METHODS Sandwich configuration was used to culture hepatocytes and the structure of bile canaliculi as well as the function of bile excretion was observed by immunocytochemistry and the test of FDA metabolism with a single collagen configuration as a control. RESULTS First, the formation of bile canaliculi was observed by immunocytochemistry. In sandwiched hepatocytes, the gradual development of bile canaliculi-like structures into an anastomotic network was observed. At 24 h after cell culture, the formation of bile canaliculi was observed. With time progressed, the bile canaliculi became more clear and the network was established at 120 h. In contrast, hepatocytes in single collagen configuration showed almost no network of bile canaliculi. Second, the ability for hepatocytes to internalize, metabolize and excrete compounds into bile was indicated by FDA metabolized in the hepatocytes. In sandwiched hepatocytes, the bile excretory function was shown at 96 h, but in single collagen system, no bile excretion was observed. CONCLUSIONS Cultured hepatocytes are able to regain bile excretion in a given certain condition. Sandwich-cultured hepatocytes can reestablish bile canalicular structure and regain bile excretory function.