Hepatic ischemia reperfusion injury (HIRI) is a pathophysiological and complex systemic process involving multiple tissues and organs. Gastrodin (GSTD), a natural compound from Gastrodia elata, displays a variety of interesting pharmacological activities. Heme oxygenase-1 (HO-1), a stress-responsive protein, has a cytoprotective defense response against oxidative and inflammatory injuries. The aim of this investigation was to elucidate whether GSTD plays a protective role against HIRI by regulating HO-1 expression. GSTD (100 mg/kg) or zinc protoporphyrin (15 mg/kg; an HO-1 inhibitor) was administered to HIRI C57 male mice. GSTD decreased glutamic pyruvic transaminase and glutamic oxaloacetic transaminase levels in HIRI mice. Inflammatory (TNF-alpha and IL-6) and oxidative-stress (malondialdehyde, MDA) markers of HIRI mice were decreased by GSTD. GSTD up-regulated HO-1 protein and mRNA expression in HIRI mice but decreased caspase-3 and -9 protein expression. GSTD lowered mRNA expression of apoptosis-related genes (caspase-3, -9, -12, and Bax) in the liver of HIRI mice but enhanced mRNA level of the anti-apoptotic Bcl-2 gene. Consistent with in vivo results, GSTD displayed a similar regulatory effect on the expression of mRNA (HO-1, caspase-3, -9, -12, Bax, and Bcl-2) and protein (HO-1, caspase-3 and -9) as well as inflammatory (TNF-alpha and IL-6) and on oxidative stress factors (superoxide dismutase and MDA) in BRL-3A cells transfected with small interfering HO-1 RNA in a hypoxia-reperfusion model. In conclusion, GSTD up-regulated HO-1 expression to play a protective role in HIRI by anti-apoptotic, anti-inflammatory, and antioxidant effects. GSTD is a promising natural compound that alleviated HIRI in liver surgery.