Doxorubicin (DOX) remains a cornerstone of chemotherapy but is limited by dose-dependent doxorubicin-induced cardiotoxicity (DIC). Recent evidence suggests that mitochondrial dysfunction, particularly oxidative stress and ferroptosis, drives DIC progression. While the flavonoid Calycosin (CAL) is known for its cardioprotective properties, the precise mechanism by which it maintains mitochondrial homeostasis remains elusive. This study aimed to investigate the effects of CAL against DIC and to explore the mechanisms by which CAL regulates mitochondrial integrity and iron homeostasis. Using both in vitro H9c2 cardiomyocytes and in vivo C57BL/6 mice, we demonstrate that CAL significantly restores cardiac contractility and reduces myocardial damage. Transcriptomic analysis indicated that CAL protected mitochondrial function and inhibited ferroptosis. Transmission Electron Microscopy (TEM) showed that CAL preserved mitochondrial structure. Assessments of the lipid peroxidation marker malondialdehyde (MDA) and superoxide dismutase (SOD) confirmed that CAL alleviated oxidative stress, while 8-OHdG staining verified its restoration of mitochondrial DNA integrity. Molecular docking identified the affinity of CAL with NRF2 and NRF1. CAL activated the NRF2/NRF1-TFAM axis both in vivo and in vitro, promoting mitochondrial biogenesis and respiratory function, while regulating iron homeostasis via FTMT and FPN. Knockdown or inhibition of NRF2/NRF1 abolished these protective effects of CAL. Our findings suggest that CAL acts as a dual-regulator of mitochondrial transcriptional machinery and iron metabolism, offering a novel therapeutic strategy to safeguard mitochondria against ferroptosis.