Abstract: Rat hepatocytes were obtained from SD rats by two step collagenase perfusion, and treated with different concentration of acetaminophen. The hepatocyte viability was measured by MTT, the activities of lactate dehydrogenase (LDH) was analyzed by spectrophotometer, and the expression of CYP 2E1 were measured by western blotting. With induction of acetaminophen, the hepatocyte viability decreased, the activity of LDH increased, the expression of CYP 2E1 increased. It demonstrated that acetaminophen could lead to hepatocyte injury when acetaminophen reached to higher dose.