The purpose of the current study was to evaluate the optimal compatibility proportion of 18α-Glycyrrhizic acid (18α-GA) and 18β-Glycyrrhizic acid (18β-GA) against carbon tetrachloride (CCl₄)-induced hepatic damage in rats, and further explored the underlying mechanism. Rats were injected with CCl₄ (0.1%, 0.3 ml/kg) once a week and were orally administrated with different proportions of 18α-, and 18β-GA daily for 4 weeks. Rats were then sacrificed and blood samples were collected for biochemical assay. Liver tissues were assessed histologically for severity of liver injury. Enzyme activities in liver homogenate were determined using commercial kits. The mRNA levels of associated proteins were evaluated by RT-PCR. The data showed that the combination of 18α-, and 18β-GA, especially at proportion of 4:6, obviously alleviated CCl₄-induced liver injury as evidenced by the improvement of liver histopathological changes, and decreased levels of ALT and AST in serum. Moreover, 18α- and 18β-GA at all proportions substantially improved glucose tolerance, and markedly reversed the decrease of SOD, MDA, and GSH, and increase of lipid markers (TG, TC, HDL, LDL) induced by CCl₄ via regulating the mRNA levels of SREBP-1c, ACC, PPAR-α, and CPT-1a. Collectively, these results suggested that 18α-GA in combination with 18β-GA, especially at proportion of 4:6, effectively reduced liver injury induced by CCl₄, comparable to the positive control silibinin, and the mechanism may be associated with reduced marker of liver oxidative stress and improvement of lipid metabolism via regulation of ACC, CTP-1A, PPARα, and SREBP1.