Berberine was one kind of Chinese herbal additive, which extracted from the stems and roots of various Berberis species, such as (Huanglian) and so on. It possessed a series of biological activities, including antioxidant, anti-inflammatory, reducing blood glucose and lipid level. In addition, it had been proved that berberine could improve lipid deposition and glucose metabolism in diabetes. Recently, berberine had been applied to aquaculture diets as an herbal feed additive in Asian area. Our laboratory conducted many vivo experiments to study the function of berberine. The current study demonstrated that berberine added in high-fat diet could mitigate oxidative stress, inhibit the apoptosis and enhance disease resistance of fish. However, it was found that the growth performance of fish fed with normal diet supplied berberine was significantly enhanced but immunity was not improved. In order to solve this problem, the study was aimed to explore the effect of different concentrations of berberine on cell activity, oxidative stress and apoptosis of grass carp () hepatopancreas cell in vitro experiment. Cultured cells were treated with different concentrations of berberine (0, 5 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L) for 0, 6 h, 12 h, 24 h. Then, the optimal concentration of berberine was showed by detecting cell viability in CCK-8 assay. Results showed that the cell activity was affected by interaction of time and berberine concentration (<0.05) affected cell activity in a dose-dependent manner. According to the cell activity data, the remaining indicators were selected to data of 12 hours for analyzing related indicators, including glutamate oxalate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activity, malondialdehyde (MDA) and ROS content, apoptosis, caspase-3, caspase-8 and caspase-9 mRNA expression. Compared with control group, GOT and GPT activity in group of 100 μmol/L berberine concentration were significantly (<0.05) increased. Meanwhile, the content of MDA and ROS were significantly increased with increasing concentration of berberine. The tendency of content of MDA and ROS were same as that of GOT and GPT activity (<0.05). In addition, compared with control group, the total cell apoptosis rate in group of 25 μmol/L and 50 μmol/L berberine concentration were significantly (<0.05) increased while cell apoptosis rate in 100 μmol/L berberine group was significantly (-3 in group of 50 μmol/L and 100 μmol/L berberine group were significantly enhanced (<0.05). In summary, the safety concentration of berberine in stimulating fish liver cells should be 25-50 μmol/L. When the concentration was more than 50 μmol/L, it would have toxicity to normal growth cells and affect cell function.