The aim of this study was to investigate the effects of various concentrations of Sargassum hemiphyllum extract on cell viability, antioxidation system in primary rat hepatocytes and hepatic protection against carbon tetrachloride (CCl4)-induced hepatic damage. The results showed that the primary rat hepatocytes had the maximum cell viability at 5.0 mg/ml and was significantly higher than negative control (p < 0.05). In addition, the treatment of S. hemiphyllum extract could increase the content of intracellular glutathione (GSH) and the activity of glutathione-S-transferase (GST) in primary rat hepatocytes. The S. hemiphyllum extract exhibited significant protective activity against CCl4 induced hepatotoxicity in primary rat hepatocytes, and restored the level of cell viability, GSH content and the activities of antioxidant enzymes such as GST, glutathione reductase (GRd), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT). Finally, the oral treatment with S. hemiphyllum extract for 8 consecutive weeks could cause significant reduction of both GOT and GPT levels after the co-administration of CCl4 induced hepatic damage in rats. It was found that S. hemiphyllum extract could significantly decrease GOT and GPT. In antioxidant activity evaluation, administration of S. hemiphyllum extract also could significantly increase the GSH content and the activity of GPx, GRd, GST and SOD in liver. The histopathological evaluation of the liver also revealed that S. hemiphyllum extract reduced the incidence of liver lesions induced by CCl4. Based on these result, we suggested that S. hemiphyllum extract exerted their hepatoprotective activities against CCl4-induced injury by preserving the cellular antioxidative defense system.