To investigate the role of knockdown of lipocalin-2 (LCN2) on DHODH mediated mitochondrial ferroptosis in sepsis-induced myocardial injury (SIMI) in mouse and cardiomyocyte injury model, and investigate the likely mechanism of STAT3. In this study, we established an sepsis induced cardiomyopathy (SIMI) model using cecal ligation and puncture (CLP) in mice. Mouse cardiac samples were used to identify the association between the serum lipocalin 2 (LCN2) level and spesis progression. Chemical inhibition of ferroptosis were conducted to illustrate the effect of ferroptosis upon SIMI.Then, mouse models with knockdown of LCN2 were used to ascertain the role of LCN2 upon ferroptosis and spesis. To interaction mechanism between LCN2 and DHODH was investigated. we overexpressed DHODH in mouse HL-1 cardiomyocytes using lentivirus, and established a lipopolysaccharide (LPS)-induced cardiomyocyte injury model. The results showed that in septic mice, the increase of LCN2 was correlated positively with myocardial severity, glutathione peroxidase 4 (GPX4) and dihydroorotate dehydrogenase (DHODH) mediated ferroptosis both occurred in SIMI. Inhibiting ferroptosis reduced LCN2 expression, thereby alleviated SIMI. Molecular docking and immunoprecipitation revealed strong binding between LCN2 and DHODH. Knockdown of LCN2 inhibited DHODH mediated ferroptosis, decreased phosphorylated STAT3 (p-STAT3) protein expression level, improved mitochondrial function, and mitigated SIMI. Meanwhile, exogenous recombinant LCN2 protein induced mouse cardiomyocyte ferroptosis. Overexpression of DHODH protected against LPS-induced cardiomyocyte injury. Inhibition of DHODH abolished knockdown-LCN2 induced decrease of p-STAT3 protein expression. However, after inhibition of mitochondrial GPX4 expression by Fin56, p-STAT3 protein was not changed. In summary, our study confirmed knockdown of LCN2 inhibited DHODH mediated ferroptosis in SIMI, inhibiting DHODH attenuated the protection of knockdown-LCN2 through inhibiting STAT3 phosphorylation. It provides the new insight for preventing sepsis-induced myocardial injury.