PURPOSE: This study aimed to explore the mechanism of Zuogui Jiangtang Shuxin formula (ZGJTSXF) in the treatment of diabetic cardiomyopathy (DCM) by an integrative strategy combining serum pharmacochemistry, network pharmacology analysis, and experimental validation. METHODS: An Ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-Exactive-Orbitrap-MS) method was constructed to identify compounds in rat serum after oral administration of ZGJTSXF. A component-target network between the targets of ZGJTSXF ingredients and DCM was established using Cytoscape. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to deduce ZGJTSXF-associated targets and pathways. The DCM model mice were treated with ZGJTSXF, and the predicted important signaling pathways were verified using quantitative PCR and Western blot. RESULTS: We identified 78 compounds in serum of medicated rats, which mainly included flavonoids, small peptides, nucleosides, organic acids, phenylpropanoids, alkaloids, phenanthrenequinones, iridoids, phenols, and saponins. Network pharmacology analysis revealed that ZGJTSXF may regulate targets including ALB, TNF, AKT1, GAPDH, VEGFA, EGFR, SRC, CASP3, MAPK3, JUN, and PI3K/AKT signaling pathway in the treatment of DCM. ZGJTSXF administration improved blood sugar levels, heart function, and cardiac morphological changes in DCM mice. Notably, ZGJTSXF inhibited cardiomyocytes apoptosis, which was associated with restored PI3K/Akt signaling and upregulated Bcl-2 and Bcl-xL proteins expression. CONCLUSION: Our preliminary results proposed the material basis and possible mechanisms of ZGJTSXF in treating DCM, which is related to the activation of the PI3K/AKT signaling pathway and apoptosis inhibition. These findings shed new light in developing ZGJTSXF-based therapeutics in treating DCM. CI - (c) 2023 Huang et al.