Thymoquinone (TQ) is the main active constituent of Nigella Sativa. The present study aimed to investigate the effect of TQ on apoptotic parameters and MMP-9 expression in isoproterenol (ISP)-induced myocardial infarction (MI). TQ was given once daily for 7 days at doses of 10 and 20 mg/kg orally with ISP (86 mg/kg; s.c.) administered on the 6th and 7th days. TQ pretreatment protected against ISP-induced MI as approved by normalization of electrocardiogram (ECG) and b (CK)-MB, minimal histopathological changes, and reduction of the infarction size. Effects of TQ could be supported by its antioxidant activity, evidenced by the increase of cardiac reduced glutathione and total serum antioxidant capacity, and the inhibition of ISO-induced lipid peroxidation. TQ anti-inflammatory activity was associated with reduced expression of NF-κB and TNF-α. TQ ameliorated cardiomyocytes, apoptotic pathways by inhibiting both the intrinsic pathway, via reducing cytoplasmic cytochrome C, and the extrinsic pathway, by inhibiting TNF-α and caspases, and the effect of TQ was dose-dependent. Moreover, TQ reduced the expression of metalloproteinase (MMP)-9, which is considered as a prognostic marker of ventricular remodeling, recommending that TQ can be used as a possible supplement to minimize post-MI changes. So, we conclude that TQ antiapoptotic activity and the inhibitory modulation of MMP-9 expression contribute to TQ protective effects in MI. Up to our knowledge, this is the first study reporting the effect of TQ on cytochrome c activity and MMP-9 expression in MI.