The aim of this study was to investigate the hepatoprotective action of the protein fraction of Phyllanthus niruri against acetaminophen (APAP) hepatotoxicity. The partially purified protein fraction of P. niruri was injected intraperitoneally in mice either prior to (preventive) or after the induction of toxicity (curative). Levels of different liver marker enzymes in serum and different antioxidant enzymes, as well as lipid peroxidation in total liver homogenates were measured in normal, control (toxicity induced) and P. niruri protein fractiontreated mice. P. niruri significantly reduced the elevated glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) levels in the sera of toxicity induced mice, compared with the control group. Lipid peroxidation levels were also reduced in mice treated with P. niruri protein fraction compared with the APAP treated control group. Among the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione–S-transferase (GST) levels were restored to almost normal levels compared with the control group. P. niruri treatment also enhanced reduced hepatic glutathione (GSH) levels caused by APAP administration. The results demonstrated that the protein fraction of P. niruri protected liver tissues against oxidative stress in mice, probably acting by increasing antioxidative defense.