Nonalcoholic fatty liver disease (NAFLD) is now the major chronic liver disease in the world. Iron overload occurring in CLDs is a risk factor for the disease progression; however, it is still unclear whether iron overload contributes to the progression of NAFLD to nonalcoholic steatohepatitis (NASH). In this study, we investigated the pathological role of iron overload in a rat NASH model. Six-week-old male F344 rats were fed a control, high-fat (HF), high-fat high-iron (HFHI) and high-iron (HI) diets for 30 weeks. Rats in HF and HFHI groups showed an ALT-dominant elevation of serum transaminases, hepatic steatosis, and inflammation with upregulation of proinflammatory cytokines. In HFHI group, the number of inflammatory foci, equivalent to lobular inflammation in NASH patients, significantly increased with upregulation of inflammatory cytokines such as TNF-α, compared to HF group. Macrophages laden with iron were seen in the inflammatory lesion of HFHI group. These results suggested that dietary iron supplementation enhances experimental steatohepatitis induced by long-term HF diet feeding. Excessive activation of macrophages stimulated by iron accumulation can be involved in the exacerbation of inflammation.