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 whic 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.