Geniposide (GEN), a medical herb, is known for its therapeutic applications in cardiovascular diseases, though its efficacy in treating myocardial ischemia/reperfusion injury (MI/RI) is yet to be fully elucidated. This study is an endeavor to explore the potential protective mechanism of GEN against MI/RI. To simulate the MI/RI condition, the left anterior descending artery was occluded for 30 min, followed by a reperfusion period of 120 min in a rat model. Three dosages (50, 100, or 150 mg/kg) of GEN were intraperitoneally injected to the Sprague-Dawley rats once a day, for seven days before the ligation of the artery. The rats were categorized into sham group, MI/RI group, and three different dosages GEN-treated groups. As the results showed, the pretreatment with GEN mitigated myocardial injury, reduced infarct volume, inhibited apoptosis, enhanced superoxide dismutase activity, and decreased malondialdehyde and myeloperoxidase activity, as well as serum creatine kinase-MB and lactate dehydrogenase levels. Moreover, GEN ameliorated MI/RI by downregulating protein expression of toll-like receptor 4, myeloid differentiation primary response 88, and p-nuclear factor-κB. In conclusion, the pretreatment of GEN may be considered as a potential therapeutic option for MI/RI.