Bacterial keratitis (BK) is a serious ocular infection that can cause severe inflammation and corneal scarring, leading to vision loss. In this study, we aimed to investigate the involvement of ferroptosis in the pathogenesis of BK.Transcriptome analysis was performed to evaluate ferroptosis-related gene expression in human BK corneas. Subsequently, the ferroptosis in mouse models of Pseudomonas aeruginosa keratitis and corneal stromal stem cells (CSSCs) were validated. The mice were treated with levofloxacin (LEV) or levofloxacin combined with ferrostatin-1 (LEV+Fer-1). CSSCs were treated with lipopolysaccharide (LPS) or LPS combined Fer-1. Inflammatory cytokines, α-SMA, and ferroptosis-related regulators were evaluated by RT-qPCR, immunostaining, and Western blot. Iron and reactive oxygen species (ROS) were measured.Transcriptome analysis revealed significant alterations in ferroptosis-related genes in human BK corneas. In the BK mouse models, the group treated with LEV+Fer-1 exhibited reduced inflammatory cytokines (MPO, TNF-α, and IFN-γ), decreased corneal scarring and α-SMA expression, and lower Fe3+ compared to the BK and LEV groups. Notably, the LEV+Fer-1 group showed elevated GPX4 and SLC7A11 in contrast to the BK and LEV group. In vitro, Fer-1 treatment effectively restored the alterations of ROS, Fe2+, GPX4, and SLC7A11 induced by LPS in CSSCs.Ferroptosis plays a crucial role in the pathogenesis of BK. The inhibition of ferroptosis holds promise for mitigating inflammation, reducing corneal scarring, and ultimately enhancing the prognosis of BK. Consequently, this study provides a potential target for innovative therapeutic strategies for BK, which holds immense potential to transform the treatment of BK.