Objective To investigate the protective effects of glutathione (GSH) against hepatic ischemia/reperfusion (I/R) injury and the related mechanism. Methods SD rats were randomized into Sham, I/R and GSH groups with 20 rats in each group. Rat models of segmental (70%) warm hepatic ischemia were established in I/R and GSH groups. GSH was injected through the femoral vein at the dose of 5 mg/kg 5 min before ischemia. At 24 h after reperfusion, liver injury was evaluated by serological and histological indices. Liver cell apoptoses were evaluated by TUNEL staining. The GSH/oxidized glutathione (GSSG) ratios of tissue level were compared between different groups. Liver mitochondria were collected and the mitochondrial calcium capacity (CRC) was evaluated. Results The serum aspartate transaminanse (AST) and alanine aminotransferase (ALT) levels in GSH group were significantly decreased 6 h after reperfusion compared with I/R group(P<0.05). 24 h after reperfusion, the liver injury was alleviated and the number of apoptosis cells was significantly decreased in GSH group compared with I/R group (P<0.05). The GSH/GSSG ratio of tissue level in GSH group was significantly increased 6 h after reperfusion compared with I/R group (P<0.05). Liver mitochondrial CRC in GSH group was significantly increased 6 h after reperfusion compared with I/R group (P<0.05). Conclusion GSH preconditioning can protect liver from hepatic I/R injury, which is possibly by inhibiting oxidative response and subsequent inhibition of mitochondrial permeability transition.