The aim of the present study was to investigate the protective effects of Shenfu injection (SFI) against myocardial ischemia–reperfusion injury (MIRI) in model rats and to explore its mechanism of action. Sprague–Dawley (SD) rats were pretreated with SFI and N ^G-nitro-L-arginine methyl ester (L-NAME) via tail vein injection and then rats were subjected to ischemia by occlusion of the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. Left ventricular function was evaluated by echocardiography. Hemodynamic was measured by the Millar pressure–volume system; serum creatine kinase (CK), lactate dehydrogenase (LDH) and serum troponin (TNNI3) levels were determined. Myocardial infarct size was observed by 2,3,5-triphenyl-2 H -tetrazolium chloride (TTC) staining; p-Akt/Akt, and p-endothelial nitric oxide synthase (p-eNOS)/eNOS levels were assessed by Western blotting; nitric oxide (NO) content in serum was determined by the Griess reaction. SFI significantly decreased serum CK, LDH and TNNI3 levels in MIRI rats, while it significantly increased the level of left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), maximal rate of the increase of left ventricular pressure (+d p /d t _max), maximal rate of the decrease of left ventricular pressure (−d p /d t _max), left ventricle ejection fraction percentage (EF), and stroke volume (SV). In addition, SFI significantly reduced myocardial infarction area and activated the phosphorylation of eNOS via Akt. The phosphorylation of eNOS and the concurrent increase of NO production contributed significantly to the protective effects of SFI. These results demonstrate that SFI protects the rat heart against MIRI and that this effect is mediated in part by Akt/eNOS signaling.