This study was designed to investigate the hepatoprotective effect of diffractaic acid isolated from a lichen species, Usnea longissima, at 3 doses, 50, 100 and 200 mg/kg, against carbon tetrachloride (CCl4)-induced hepatic damage. For this purpose, 40 Wistar albino rats were divided in 5 groups, including 3 experimental and 2 control ones, and 0.2 mL/kg of CCl4 in olive oil (1:5 v/v) was injected daily for 6 weeks intraperitoneally. After the liver injury, diffractaic acid was applied at doses of 50, 100 and 200 mg/kg for 7 days. The group given physiologic saline (0.2 mL/kg) was used as a control group. Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, creatinine, urea, direct and total bilirubin and C-reactive protein levels were also evaluated in the serum samples obtained from the rat groups. Liver tissues were removed and were examined histopathologically following staining with hematoxylin-eosin. The results showed that 50 mg/kg daily dose of diffractaic acid could be considered to have hepatoprotective effect by ameliorating the studied biochemical parameters and tissue histological structures. However, 100 and 200 mg/kg of diffractaic acid acted as hepatotoxin and did not show any hepatoprotective effect. Thus, diffractaic acid could be potentially used as a hepatoprotective agent at a low dose (50 mg/kg) against acute liver toxicity induced by CCl4.