Objective To investigate the protective effect of pirinixic acid (Wy-14643) on myocardial ischemia/reperfusion injury (MI/RI) in rats and its underlying mechanism. Methods Forty rats were randomly divided into 5 groups, with 8 rats in each group:sham operation group (sham group), ischemia-reperfusion group (MI/RI group), and low-, middle- and high-dose pirinixic acid groups (W1, W2, W3 groups, at a concentration of 1,4 and 8 mg/kg, respectively). MI/RI model was established by ligation and reperfusion of anterior descending coronary artery. Serum creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Myocardial infarction area was measured and pathological changes of myocardial tissue were observed under a light microscope. The protein expression of toll-like receptor 4(TLR4) and nuclear factor-κBp65 in myocardium was detected with Western blotting. SPSS statistics 23.0 was used for statistical analysis. Data comparison between two groups was perfomed using ANOVA analysis and Bonferroni test depending on data type. Results Serum contents of CK-MB [(94.81±8.41), (82.41±7.20) and (69.40±6.52) vs (119.91±12.71) U/L], LDH [(750.73±86.44), (654.94±79.25) and (565.26±86.00) vs (911.48±87.15) U/L], IL-6 [(176.75±11.89), (162.67±8.18) and (146.13±10.48) vs (194.93±13.91) pg/ml] and TNF-α [(80.67±7.38), (69.67±5.15) and (51.22±7.00) vs (90.44±6.68) pg/ml] were significantly lower, the area of myocardial infarction (28.00%, 16.93% and 8.38% vs 35.88%) was obviously smaller, and the expression levels of TLR4 [(1.00±0.03), (0.92±0.03) and (0.69±0.04) vs (1.33±0.03)] and nuclear factor-κBp65 [(1.01±0.03), (0.94±0.03) and (0.72±0.04) vs (1.12±0.04)] were down-regulated in the W1, W2, W3 groups when compared with the MI/RI group (P<0.05). Conclusion Preconditioning with pirinixic acid protects MI/RI myocardium by inhibiting inflammation mediated by the nuclear factor-κB signaling pathway.