Myocardial infarction (MI) remains one of the major causes of mortality around the world. A possible mechanism involved in myocardial infarction is the engagement of Toll-like receptors (TLRs). This study was intended to discover the prospective cardioprotective actions of β-caryophyllene, a natural sesquiterpene, to ameliorate isoproterenol (ISO)-induced myocardial infarction through HSP-60/TLR/MyD88/NF_B pathway. β-Caryophyllene (100 or 200mg/kg/day orally) was administered for 21 days then MI was induced via ISO (85 mg/kg, subcutaneous) on 20th and 21st days. The results indicated that ISO induced a significant infarcted area associated with several alterations in the electrocardiogram (ECG) and blood pressure (BP) indices and caused an increase in numerous cardiac indicators such as creatine phosphokinase (CPK), creatine kinase-myocardial bound (CK-MB), lactate dehydrogenase (LDH), and cardiac tropinine T (cTnT). In addition, ISO significantly amplified heat shock protein 60 (HSP-60) and other inflammatory markers, such as TNF-α, IL-Iβ, and NFkB, and affected TLR2 and TLR4 expression and their adaptor proteins; Myeloid differentiation primary response 88 (MYD88), and TIR-domain-containing adapter-inducing interferon-α (TRIF). On the other hand, consumption of β-caryophyllene significantly reversed the infarcted size, ECG and BP alterations ameliorated the ISO elevation in cardiac indicators; it also notably diminished HSP-60, and subsequently TLR2, TLR4, MYD88, and TRIF expression, with a substantial reduction in inflammatory mediator levels. This study revealed the cardioprotective effect of β-caryophyllene against MI through inhibiting HSP-60/TLR/MyD88/NFkB signaling pathways.