ABSTRACT The aim of this study was to evaluate the hepatoprotective role of a 43-kD protein (Hp-P) isolated from the leaves of Cajanus indicus L. against acetaminophen (APAP)-induced toxicity in mouse liver and in isolated hepatocytes. The hepatotoxicity of APAP and the hepatoprotective activity of Hp-P in vivo were determined by measuring the liver-specific serum marker enzymes alanine amino transferase (ALT) and alkaline phosphatase (ALP) in murine sera and observing the histological changes in the mice liver treated with the protein before and after (2 mg/kg body weight for 5 days) APAP (at a dose of 300 mg/kg body weight for 2 days) administration. The cell viability, LDH leakage, GSH level, and lipid peroxidation were measured in isolated hepatocytes to evaluate the cytotoxic effect of APAP and the protective role of Hp-P in vitro. Experimental results showed that APAP induced hepatotoxicity in vivo as revealed from the changes in serum-specific marker enzyme levels and histology of liver. It also induced cytotoxicity in hepatocytes as observed from the changes in cell viability and LDH leakage. Pretreatment with Hp-P prevented the APAP-induced elevation of ALT and ALP in murine sera. In addition, posttreatment with Hp-P significantly altered most of the changes induced by APAP. Although some natural recovery has been observed in toxin controls, the Hp-P-induced recovering process is more rapid than the natural ones. In histological studies, less centrilobular necrosis was found in the liver treated with Hp-P before and after APAP intoxication compared to the liver treated with APAP alone. Radical scavenging experiment showed that Hp-P scavenges DPPH radicals directly. Studies also showed that APAP-induced reduced cell viability and cellular LDH leakage could be prevented by the combinatorial effect of Hp-P. Besides, treatment of hepatocytes with Hp-P and APAP together maintained the normal GSH level. APAP-induced enhanced lipid peroxidation was also decreased when cells were treated with APAP and Hp-P together. Hp-P alone, on the other hand, did not induce any alterations of the studied parameters. Results of this study have been compared with a known antioxidant, alpha-tocopherol. Data from both the in vivo (before and after APAP administration) and in vitro studies suggest that Hp-P has potent hepato- and cytoprotective properties against APAP-induced toxicity.