Ruminal dysbiosis-induced liver injury is prevalent in dairy cows, yet its underlying mechanisms remain incompletely understood. Ferroptosis, a newly identified form of programmed cell death distinct from apoptosis and necrosis, has been implicated in various liver diseases by emerging studies. In the present study, lipopolysaccharide (LPS) and γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) were employed to establish in vitro and in vivo models of liver injury using bovine hepatocytes and mice, respectively. It was observed that noncytotoxic iE-DAP alone did not influence lipid peroxidation or GPX4, but exacerbated LPS-induced ferroptosis and hepatocyte injury. Notably, co-treatment with LPS and iE-DAP (LPS/iE-DAP)-induced hepatocyte injury was mitigated by intervention with the ferroptosis inhibitor ferrostatin-1 (Fer-1). Mechanistically, the activated IL-6/STAT3 signaling pathway was found to mediate LPS/iE-DAP-induced ferroptosis. Suppression of IL-6/STAT3, either through IL6 and STAT3 knockdown or pharmacological intervention, reduced Fe