The hepatoprotective efficacy of irradiated hyaluronic acid (HA) on acetaminophen (APAP) induced acute hepatotoxicity was investigated. BALB/c mice (4-6 weeks of age) were pretreated with unirradiated HA (UIHA), 5 and 50 kGy gamma irradiated HA (GIHA) for 14 days and were dosed APAP (500 mg/kg b.wt). After 9h of APAP dosing animals were euthanized. The degree of acute hepatotoxicity was measured by aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The expression of interferon-gamma (IFN-gamma) in serum and alpha-and mu-class of gluthathione-S-transferase (GSTs), CYP 2E1 class of cytochrome monooxygenase and glutathione (GSH) in liver were quantified. Histological evaluation was done by Hematoxiylin and Eiosin staining, Periodic acid schiffs staining, Manson trichrome staining and histological scorings were done. The degree of acute hepatotoxicity was markedly lower in UIHA and 5 kGy than in 50 kGy GIHA pretreated group and there was negligible difference between 5 and 50 kGy GIHA pretreated group. The expression of interferon-gamma (IFN-gamma) was significantly (P<0.05) suppressed in 5 and 50 kGy GIHA pretreated group. Histological scorings showed a significant protection of liver in UIHA and 5 kGy GIHA pretreated mice. Expression of alpha class GSTs was significantly increased in 5 and 50 kGy GIHA pretreated group. To conclude suppression of IFN-gamma and increase in alpha-class GSTs expression may exert a protective role in acute hepatotoxicity of APAP and 5 kGy GIHA showed comparable protective effect to that of UIHA.