BACKGROUND: Doxorubicin (DOX), a widely used anthracycline chemotherapy agent, is effective against various malignant tumors. However, its clinical application is significantly limited due to dose-dependent cardiotoxicity. Linggui Zhugan Decoction (LGZGD), a traditional Chinese medicine formulation, has demonstrated notable cardioprotective effects. However, its potential to mitigate DOX-induced cardiotoxicity (DIC) remains unexplored. OBJECTIVE: This study investigated the protective effects of LGZGD against DIC and explores its ability to enhance mitochondrial function by modulating the AMPK-FOXO3a pathway via targeting BTG2. METHODS: A zebrafish DIC model was established to evaluate the cardioprotective effects of LGZGD on embryos and adults. Further investigations included in vitro studies with H9c2 cells and in vivo experiments using mouse models to assess LGZGD's pharmacological actions and their impact on mitochondrial function. Network pharmacology and transcriptomic analyses were performed to predict the potential mechanism of LGZGD in regulating the AMPK-FOXO3a pathway via BTG2. Verification was conducted through molecular docking, molecular dynamics (MD) simulations, and immunofluorescence co-localization. RESULTS: LGZGD enhanced survival rates and alleviated heart tissue damage in zebrafish. In vitro, LGZGD reduced DOX-induced reactive oxygen species (ROS) production in H9c2 cells, decreased apoptosis, improved mitochondrial membrane potential, and preserved mitochondrial function. In vivo, LGZGD improved cardiac function and prevented myocardial structural damage in mice. Additionally, it mitigated oxidative stress, inflammation, and apoptosis while reversing DOX-induced mitochondrial structural damage. Network pharmacology and transcriptomic analyses suggested that LGZGD regulates the BTG2 gene and AMPK-FOXO3a pathway activity. Molecular docking, MDs, and immunofluorescence co-localization supported the hypothesis that LGZGD modulates the AMPK-FOXO3a pathway by targeting BTG2. CONCLUSION: LGZGD exerts significant cardioprotective effects against DIC by reducing oxidative stress, inflammation, and apoptosis preserving while mitochondrial structure and function. These findings offer a novel insight into LGZGD's clinical relevance in DIC management. Targeting BTG2 to regulate the AMPK-FOXO3a pathway highlights LGZGD as a promising therapeutic strategy for preventing and treating DIC.