Background: Myocardial ischemia (MI) is a leading cause of death worldwide. Menispermi Rhizoma is a traditional Chinese medicine that exerts a variety of beneficial pharmacological activities in many diseases, including MI.Purpose: Serum pharmacochemistry and network pharmacology were used to explore the material basis and mechanism of action of Menispermi Rhizoma against MI.Methods: The absorbed components of Menispermi Rhizoma in rat plasma were analyzed by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). The key components, targets, pathways, and interrelated information were obtained by network pharmacology. The potential effective components of Menispermi Rhizoma against MI were screened by methyl-thiazolyl-tetrazolium (MTT) assay, and the cardioprotective effect and mechanism of active components were verified by Western blotting and molecular docking.Results: In total, 25 absorbed components of Menispermi Rhizoma in plasma were identified. Network pharmacology revealed 81 major targets of Menispermi Rhizoma against MI, mainly involving the regulation of the PI3K/AKT and MAPK pathways. In vitro validation of H9c2 cells revealed that acutumine, daurisoline, dauricoside, and 6-O-demethylmenisporphine are the main bioactive components of Menispermi Rhizoma. The levels of lactate dehydrogenase, creatine kinase, and malondialdehyde (MDA) were significantly decreased by four alkaloids, whereas the activities of superoxide dismutase (SOD) and glutathione (GSH) were significantly increased. Four alkaloids effectively protected H9c2 cells against OGD-induced apoptosis by Hoechst/PI staining and flow cytometry assay. Western blotting results showed that the four alkaloids upregulated the expression ratio of Bcl-2/Bax and downregulated the expression levels of Cyt-C and cleaved caspase 3, which further supported the anti-cardiomyocyte apoptosis and antioxidative stress effect of Menispermi Rhizoma. Molecular docking confirmed that the four compounds were capable of binding to AKT1, MAPK1, EGFR, CASP3, and MAPK8 proteins, suggesting the protective effect of Menispermi Rhizoma on MI via PI3K/AKT, MAPK, and apoptosis pathways.Conclusion: Menispermi Rhizoma exerted cardioprotective effects through the effect characteristics: multiple-ingredient, multi-target, and multi-pathway. This research provided a reference for further mechanistic research on wider applications of Menispermi Rhizoma for MI treatment.