Abstract: Objective: Determining the characteristics of human fetal hepatocytes in vitro. Methods: Isolating and culturing human fetal hepatocytes by stepwise trypsinization of liver fragment in vitro; collecting the culture medium to determine the quantity of AFP, ALB and the functional enzymes (including ALT, AST, GGT, ALP and LDH) of different generations in culture; determining the expression of cytochrome C by immunohistochemistry; testing the effects of sodium byturate on human fetal hepatocytes. Results: Human fetal hepatocytes were polygonal epithelial cells in DMEM medium. They could be maintained for 5.5 months (about 30 passages) in vitro. They secreted ALB and functional enzymes all over their cultivation. Conclusion: Human fetal hepatocytes can be maintained and keep function in vitro for several months.