Objective To investigate the protective effect of Scutellaria barbata flavonoids (SBF) on myocardial ischemia/reperfusion injury (MI/RI) model and its underlying mechanism. Methods Fifty female SD rats randomly divided into 5 groups, with 10 rats in each group, that is, sham operation group (sham group), MI/RI group, and low-, middle- and high-dose SBF groups (30,75 and 140 mg/kg SBF respectively). The rat MI/RI models were established by ligating left anterior descending coronary arteries. Serum contents of creatine kinase isoenzyme-MB (CK-MB) and lactate dehydrogenase (LDH) were detected by enzyme linked immunosorbent assay (ELISA). The myocardial infarct area was measured using NBT staining. The protein levels of p53, Caspase-3, B-cell lymphoma-2 (Bcl-2) were detected by Western blot. The apoptosis rate of cardiomyocytes was measured by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL). SPSS statistics 25.0 software was used for statistical analysis. One-way ANOVA was employed for inter-group comparisons. Results Compared with the MI/RI group, the low-, middle-and high-dose groups had obviously decreased serum contents of LDH [(807.51±30.74) vs (724.24±44.88) vs (568.63±40.49) vs (448.52±49.67) U/L] and CK-MB [(106.58±7.99) vs (92.45±4.88) vs (79.47±6.71) vs (70.51±5.88) U/L], protein levels of p53 [(1.19±0.05) vs (1.06±0.03) vs (1.02±0.03) vs (0.77±0.03)] and Caspase-3 (1.50±0.05) vs (1.01±0.05) vs (0.96±0.04) vs (0.41±0.04)], area of myocardial infarct (37.21% vs 27.45% vs 16.08% vs 8.00%) and cardiomyocytes apoptosis rates (43.82% vs 35.91% vs 32.01% vs 22.67%) (P<0.01, P<0.05), but significantly increased expression levels of Bcl-2 [(1.02±0.04) vs (1.37±0.08) vs (1.42±0.04) vs (1.62±0.04)] (P<0.01). Conclusion SBF preconditioning can inhibit the apoptosis pathway induced by p53 signaling pathway, and play a protective effect against MI/RI.