The effects of intravenous high mobility group box 1 (HMGB1) on myocardial ischemia/reperfusion (I/R) injury remains to be elucidated. The purpose of the present study was to investigate the effects of intravenous HMGB1 on the expression of hypoxia inducible factor-1α (HIF-1α) in the myocardium of rats following acute myocardial ischemia, and to examine the effects of intravenous HMGB1 on myocardial I/R injury. Male Wistar rats were divided into the following groups: Sham operation group (n=10), a group exposed to ischemia for 30 min and reperfusion for 4 h (I/R group) as a control (n=10), an HMGB group, in which 100 ng/kg HMGB was administered intravenously 30 min prior to ischemia (n=10), an LY group, in which LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), was administered intravenously (0.3 mg/kg) 40 min prior to ischemia (n=10), and the HMGB1+LY group, in which HMGB1 (100 ng/kg) and LY294002 (0.3 mg/kg) were administered intravenously 30 min and 40 min prior to ischemia, respectively (n=10). The serum levels of cardiac troponin I (cTnI) and tumor necrosis factor-α (TNF-α), and myocardial infarct size were measured. The expression levels of phosphorylated Akt and HIF-1α were investigated using western blot analyses. The results showed that pre-treatment with HMGB1 significantly decreased serum levels of cTnI, and TNF-α, and reduced myocardial infarct size following 4 h reperfusion (all P<0.05). HMGB1 also increased the expression levels of HIF-1α and p-Akt induced by I/R (P<0.05). LY294002 was found to eliminate the effects of intravenous HMGB1 on myocardial I/R injury (P<0.05). These results suggest that intravenous pre-treatment with HMGB1 may exert its cardioprotective effects via the upregulation of the myocardial expression of HIF-1α, which may be regulated by the PI3K/Akt signaling pathway, in rats following acute myocardial I/R.