Thymus quinquelostatus Celak, a kind of food additive and folk medicine, has obvious antioxidative and anti-inflammatory effects. However, there have been no reports about the cardioprotective effects of polyphenol-rich fraction from Thymus quinquelostatus Celak (TCPRF). Here, we predicted the targets and pathways of TCPRF related to myocardial ischemia (MI) using network pharmacology, and examined the effects as well as the mechanisms of TCPRF in ISO-induced MI mice. The results verified that TCPRF could decrease the serum CK level, improve ECG and the pathological alterations of myocardium. TCPRF also increased SOD activity, decreased MDA content, reduced the positive apoptotic cells, decreased the expression of Bax, and increase the expression of Bcl-2, PI3K and p-AKT in the myocardium. These results demonstrated that TCPRF was able to alleviate ISO-induced MI injury, which is partly related to inhibiting myocardium apoptosis, antioxidation and activating PI3K/AKT pathway.