Berberine, an isoquinoline alkaloid originally isolated from the Chinese herb Coptis chinensis (Huanglian), has been shown to display a wide array of pharmacological activities. The present study was to investigate the effects of berberine against myocardial ischemia produced in rats by isoproterenol. 50 male Sprague-Dawley rats were randomized equally into five groups: a control group, an untreated model group, berberine (30, 60 mg/kg) treatment, or propranolol (30 mg/kg). Rats were treated for 12 days and then given isoproterenol, 85 mg/kg for 2 consecutive days by subcutaneous injection. ST-segment elevation was measured after the last administration. Serum levels of creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured after the rats were sacrificed. The hearts were excised for determining heart weight index, microscopic examination, high mobility group box 1 (HMGB1), toll-like receptor (TLR4), prodeath protein (Bax), antideath protein (Bcl-2), and tumor necrosis factor (TNF-α) protein were determined by western blot. Berberine decreased the ST elevation induced by acute myocardial ischemia, and decreased serum levels of CK-MB, LDH, TNF-α, and IL-6. Berberine increased total superoxide dismutase (T-SOD) activity and decreased malondialdehyde (MDA) content in myocardial tissue. Berberine can regulate HMGB1-TLR4 axis to protect myocardial ischemia.