To investigate the effect of different concentrations of matrine on the human hepatocytes in vitro. The human hepatocytes were cultured respectively at 250mg/L, 500mg/L and 1000mg/L matrine for 6 days, the morphological changes of cells were observed. The alive cell number was detected by MTT assay and BCA assay. Content of LDH (lactate dehydrogenate) was measured to find out the integrity of the cell membrane. The cells cultured with 500mg/L and 1000mg/L matrine appeared distinct morphological changes and many dead hepatocytes were found in the sixth day. The values of MTT assay decreased in the fourth day of 1000mg/L group and in the sixth day of 500mg/L and 1000mg/L group, was statictically significant compared to that in the control group (P<0.05). The protein quantities of cells in the fourth and sixth days of 1000mg/L group, were significantly different compared to the control group (P<0.05). The value of LDH increased in the second and sixth day of 500mg/L group, and in the second, the fourth and the sixth days of 1000mg/L group, which was statistically significant compared to the control group(P<0.05). When the concentration of matrine was 500mg/L～1000mg/L, it could damage human hepatocytes in vitro obviously.