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Excessive alcohol and iron intake can reportedly cause liver damage. In the present study, we investigated the effect of Lactobacillus casei on liver injury in rats co‐exposed to alcohol and iron and evaluated its possible mechanism. Sixty male Wistar rats were randomly divided into three groups for 12 weeks: the Control group (administered normal saline by gavage and provided a normal diet); alcohol +iron group (Model group, treated with alcohol [3.5–5.3 g/kg/day] by gavage and dietary iron [1,500 mg/kg]); Model group supplemented with L. casei (8 × 108 CFU kg−1 day−1) (L. casei group). Using hematoxylin and eosin (HE) staining and transmission electron microscopy, we observed that L. casei supplementation could alleviate disorders associated with lipid metabolism, inflammation, and intestinal mucosal barrier injury. Moreover, levels of serum alanine aminotransferase, gamma‐glutamyl transferase, triglyceride (TG), and hepatic TG were significantly increased in the model group; however, these levels were significantly decreased following the 12‐week L. casei supplementation. In addition, we observed notable improvements in intestinal mucosal barrier function and alterations in T lymphocyte subsets and natural killer cells in L. casei‐treated rats when compared with the model group. Furthermore, L. casei intervention alleviated serum levels of tumor necrosis factor‐α and interleukin‐1β, accompanied by decreased serum endotoxin levels and downregulated expression of toll‐like receptor 4 and its related molecules MyD88, nuclear factor kappa‐B p65, and TNF‐α. Accordingly, supplementation with L. casei could effectively improve liver injury induced by the synergistic interaction between alcohol and iron. The underlying mechanism for this improvement may be related to immune regulation and inhibition of enterogenic endotoxin‐mediated inflammation. [ABSTRACT FROM AUTHOR]