Background: Oxytocin (OT) has shown a cardioprotective effect on myocardial ischemia/reperfusion injury (MIRI). This study aimed to investigate whether the cardioprotective effect of OT is associated with the inhibition of mast cell degranulation and inflammation. Methods: The left anterior descending coronary artery of rats was ligated for 30 min and reperfused for 120 min to establish an ischemia and reperfusion (I/R) injury model. A preliminary experiment was conducted to evaluate the optimal dose of OT (0.01, 0.1, 1 μg/kg via intraperitoneal). The mast cell secretagogue compound 48/80 (C48/80) was used to promote the degranulation of mast cells with or without I/R injury, while rats were pretreated with OT to determine whether this compound suppresses mast cell degranulation. The expression of the inflammatory factors HMGB1 and NF-κB p65 was evaluated. A cell experiment was performed for verification. Results: C48/80 (0.5 mg/kg, intravenous) increased mast cell degranulation and tryptase release compared with I/R-treated alone (27.12 ± 3.52 % vs. 16.57 ± 2.23 %; 8.34 ± 1.66 ng/mL vs. 3.63 ± 0.63 ng/mL), but these effects could be decreased by OT (0.1 μg/kg, intraperitoneal) preconditioning (19.29 ± 0.74 %; 5.37 ± 0.73 ng/mL). Besides that, hemodynamic disorders, arrhythmias, cardiac edema, infarct size, histopathological damage, and the levels of cTnI, HMGB1 and NF-κB p65 were significantly increased in I/R-treated group compared with corresponding observations in the control group, and C48/80 exacerbated these injuries, but pretreatment with OT could ameliorate these effects. Furthermore, C48/80 (10 μg/mL) inhibited the viability and promoted the apoptosis of H9C2(2-1) and RBL-2H3 cells, and increased the release of cTnI and tryptase, all of which were reversed by prophylactic OT (0.01 ng/mL) treatment. Conclusion: We concluded that OT pretreatment inhibits the degranulation of cardiac mast cells induced by I/R injury and downregulates the expression of the inflammatory factors HMGB1 and NF-κB p65. © 2020 The Authors