Oxidative stress is a leading cause to liver injury. Rac2 is a Ras-associated guanosine triphosphatase, an important molecule modulating a large number of cells and involved in the regulation of reactive oxygen species (ROS). For the study described here, we supposed that Rac2 knockout protects mice against CCl₄-induced acute liver injury. We found that Rac2 expressed highly in CCl₄-induced liver tissues. CCl₄-treated Rac2 knockout (Rac2−/−) mice had reduced CD24 levels and steatosis. In addition, CCl₄-induced high expression of pro-inflammatory cytokines and chemokine were reversed by Rac2 deficiency compared to CCl₄-treated wild type (WT) mice. We also found that fibrosis-related signals of MMP-9, MMP-2 and TGF-β1 were also down-regulated in Rac2 knockout mice induced by CCl₄. Significantly, oxidative stress induced by CCl₄ was also suppressed owing to the lack of Rac2, evidenced by enhanced superoxide dismutase (SOD) activity, and reduced malondialdehyde (MDA) levels, superoxide radical, H₂O₂, xanthine oxidase (XO), xanthine dehydrogenase (XDH) and XO/XDH ratio. Moreover, c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPK) was activated by CCl₄, which was reversed in the liver of Rac2−/− mice through western blot and immunohistochemical analysis. In vitro, endotoxin (LPS) was treated to hepatocytes isolated from WT mice and Rac2−/− mice. The data further confirmed the role of Rac2 deficiency suppressed pro-inflammatory cytokines and chemokine, as well as fibrosis-related signals. Of note, production of ROS induced by LPS was reduced in Rac2−/− cells, accompanied with enhanced SOD1, SOD2 and reduced XO and phosphorylated-JNK expressions. Our results indicated that Rac2 played an essential role in acute liver injury induced by CCl₄, providing the compelling information of the effects of Rac2 on liver injury, and revealing a novel regulatory mechanism for acute liver injury.