Metformin exerts antidiabetic and pleiotropic effects. This study investigated the function and mechanisms of gasotransmitters and autophagy in the metformin-induced protection against ischemia/reperfusion injury (I/RI). According to measurements of serum hepatic function indicators and histopathological evaluation, metformin protected against hepatic I/RI-induced impairment of liver function and structure. In addition, metformin inhibited hepatic I/RI-induced hepatic oxidative stress, nitrosative stress, inflammation, and apoptosis. Also, it suppressed hepatic I/RI-induced decrease in hepatic heme oxygenase-1 (HO-1) and hydrogen sulfide (H2S) levels and increase in nitric oxide (NO) production. Furthermore, metformin inhibited hepatic I/RI-induced decrease in protein expressions of endothelial NO synthase (eNOS), HO-1, cystathionine γ-lyase (CSE), and Beclin-1 and increase in the protein expression of inducible NO synthase (iNOS) in the liver tissue. Co-administration of the NO biosynthesis inhibitor, L-NAME, carbon monoxide(CO)-releasing molecule-A1 (CORM-A1), the H2S donor, NaHS, or the autophagy stimulator, rapamycin (RAPA), enhanced all effects of metformin. The NO donor, L-arginine, the CO biosynthesis inhibitor, zinc protoporphyrin, the H2S biosynthesis inhibitor, DL-propargylglycine, or the autophagy inhibi tor, chloroquine (CQ), antagonized the effects of metformin. These findings reveal, for the first time, that increasing CO, H2S, and autophagy levels with subsequent decreasing NO level play a critical role in metformin's protective action against hepatic I/RI. The ability of L-NAME, CORM-A1, NaHS, and RAPA to boost metformin’s protective effect in hepatic I/RI may positively be attributed to their ability to lower hepatic oxidative stress, nitrosative stress, inflammation, and apoptosis.