Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types and dosages of reagents,transferrin,epidermal growth factor and nicotinamide were used to culture porcine hepatocytes in serum-free Dulbecco’s modified Eagle’s medium (DMEM). And the proliferative effects and functions of the cells were detected at different culture times. Results Transferrin at 5 ng/ml,nicotinamide at 10 mmol/L and epidermal growth factor at 10 ng/ml had better effects on the viability of hepatocytes than DMEM,DMEM+10% fetal cattle serum FCS and other dosages of these reagents ( P <0.05). OD values of MTS were still high in culture at day 7 and day 10,while nearly 30%-35% cells went into the S phase. Good hepatocyte funcitons were found in these groups,and the secretion of albumin was positively correlated with OD value of MTS. The levels of aspartate transaminase and ammonia in these media were lower than those in DMEM and DMEM+FCS.Conclusion Transferrin at 5 ng/ml,epidermal growth factor at 10 ng/ml and nicotinamide at 10 mmol/L are benefitial to the viability and proliferation of hepatocytes. Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. -free Dulbecco’s modified Eagle’s medium (DMEM). And the proliferative effects and functions of the cells were detected at different culture times. Results Transferrin at 5 ng / ml, nicotinamide at 10 mmol / L and epidermal growth factor at 10 ng / ml had better effects on the viability of hepatocytes than DMEM, DMEM + 10% fetal cattle serum FCS and other dosages of these reagents (P <0.05). OD values ​​of MTS were still high in culture at day 7 and day 10, while nearly 30% -35% cells went into the S phase. Good hepatocyte funcitons were found in these groups, and the secretion of albumin was positively correlated with OD value of MTS. The levels of aspar tate transaminase and ammonia in these media were lower than those in DMEM and DMEM + FCS.Conclusion Transferrin at 5 ng / ml, epidermal growth factor at 10 ng / ml and nicotinamide at 10 mmol / L are benefitial to the viability and proliferation of hepatocytes .