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Guizhi Gancao Decoction (GGD) is a well-known traditional Chinese herbal medicine for the treatment of various cardiovascular diseases, such as myocardial ischemia-reperfusion (I/R) injury and arrhythmia. However, the mechanism by which GGD contributes to the amelioration of cardiac injury remains unclear. The aim of this study was to investigate the potential protective role of GGD against myocardial I/R injury and its possible mechanism. Consistent with the effect of the positive drug (Trimetazidine, TMZ), we subsequently validated that GGD could ameliorate myocardial I/R injury as evidenced by histopathological examination and triphenyltetrazolium chloride (TTC) staining. Moreover, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated that GGD suppressed myocardial apoptosis, which may be related to the upregulation of Bcl-2, PPARα, and PPARγ and downregulation of Bax, caspase-3, and caspase-9. Pretreatment with GGD attenuated the levels of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin- (IL-) 6, and IL-1β in serum by inhibiting Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. These results indicated that GGD exhibits cardioprotective effects on myocardial I/R injury through inhibition of the TLR4/NF-κB signaling pathway, which led to reduced inflammatory response and the subsequent cardiomyocyte apoptosis.