Reperfusion following liver ischemia results in oxidative stress leading to liver injury. The aim of this study was to investigate the combined effects of two antioxidant agents, rutin and L-arginine, in rat liver ischemia-reperfusion (I/R). Male Wistar rats were divided into five groups: a) sham operated, b) I/R, c) I/R + rutin, d) I/R + L-arginine, e) I/R + rutin + L-arginine. Plasmatic and hepatic levels of alanine transaminase (ALT), aspartate transaminase (AST), lipid peroxides (LOOH), thiol groups (RSH), DNA fragmentation and liver histopathology were examined. Furthermore, in order to elucidate the pathophysiological processes involved in the antioxidant mechanism/s of rutin and L-arginine we assessed the expression of nitric oxide synthase isoforms (iNOS and eNOS) and heme-oxygenase-1 (HO-1), both playing key roles in the biochemical cascade of liver injury. Significant increase in plasmatic ALT and AST activities were observed in untreated I/R rats when compared to sham-operated animals whereas treatment with rutin or L-arginine in I/R rats reduces hepatic damage. Interestingly, combined therapy with rutin and L-arginine resulted in a further reduction of plasmatic ALT and AST activities when compared to rutin or L-arginine alone. These results were further confirmed by the analysis of DNA fragmentation, LOOH, RSH groups and liver histopathology which showed the highest protective effects following the coadministration of rutin and L-arginine. Finally, the combined therapy protocol resulted in a significant induction of liver HO-1 and in a concomitant reduction of iNOS expression which may be both responsible for the beneficial effects of the proposed pharmacological protocol.