Carbon tetrachloride (CCl4)-induced liver injury causes necrosis and fibrosis, which may lead to cirrhosis and liver failure. Although a lot of study to understand the mechanism of liver fibrosis, our knowledge of the molecular level of this disease is still incomplete. The aim of this study was to investigate the effect and regulation of nitric oxide (NO) and glial fibrillary acidic protein (GFAP) on the severity of hepatic injury in male Wistar albino rats with CCl4-induced hepatotoxicity and elucidate the underlying mechanism. A total of 20 male rats were randomly divided into healthy control group (A) and CCl4-induced hepatic fibrosis model group (B), with 10 rats in each group. The results of this study suggest CCl4-induced hepatic injury is mediated by excessive NO production and up-regulated by inducible and endothelial NO synthase (P<0.05), which may plays a role in increasing hepatic injury. Additionally, liver injury induced by CCl4 demonstrated significant increases in GFAP (P<0.05). Histological and immunohistochemical analyses of the CCl4-treated group exhibited increased inflammatory process and liver necrosis/fibrosis. Inflammatory response especially NO and GFAP expression is identified to play an important role in the development of liver injury and fibrosis induced by CCl4.