Objective This study aims to determine whether over‑expression of angiotensin converting enzyme 2 (ACE2) could provide cardio‑protection against myocardial ischemia/reperfusion injury (MI/RI) through vagus nerve by mediating high mobility group box 1 (HMGB1) in rats. Methods Fifty male SD rats, weighed 220‒280 g, were randomly divided into five groups (n=10): sham operation (group S), myocardial ischemia/reperfusion injury (group I/R), unilateral vagotomy (VG)+MI/RI (group V), ACE2 overexpression+MI/RI (group A), VG+ACE2 overexpression+I/R (group VA). ACE2 overexpression model was conducted by intrapericardial injection with a trans‑diaphragmatic approach 3 weeks before the MI/RI operation, which was established with ligation of left anterior descending coronary artery for 30 min followed by reperfusion for 24 h. The infarction size was measured using Evans blue‑2,3,5‑triphenyl tetrazolium chloride staining. The protein expression of HMGB1 in myocardium was assayed with Western blot. The mRNA levels of tumor necrosis factor‑α (TNF‑α) and interleukin (IL‑6) were measured by real‑time quantitative PCR (qPCR). The serum level of cardiac troponin I (cTnI) was measured using enzyme‑linked immunosorbent assay (ELISA). Results Compared with group I/R, the infarct size, the protein level of HMGB1, the mRNA levels of TNF‑α and IL‑6 and the serum level of cTnI were significantly decreased in group A (P<0.05). Compared with group A, these parameters were significantly increased in group VA (P<0.05). Conclusions ACE2 over‑expression could provide cardio‑protection against MI/RI through vagus nerve by mediating HMGB1 in rats.