Nonalcoholic fatty liver disease (NAFLD) is now the most common liver disease in the world. NAFLD can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and eventually hepatocellular carcinoma. Acquired hepatic iron overload is seen in a number of patients with NAFLD; however, its significance in the pathology of NAFLD is still debated. Here, we investigated the role of dietary iron supplementation in experimental steatohepatitis in rats. Rats were fed a control, high-fat (HF), high-fat high-iron (HFHI) and high-iron (HI) diet for 30 weeks. Blood biochemical, histopathological and gut microbiota analyses were performed. Rats in HF and HFHI groups showed an ALT-dominant elevation of serum transaminases, hepatic steatosis, hepatic inflammation, and upregulation of proinflammatory cytokines. The number of large inflammatory foci, corresponding to lobular inflammation in NASH patients, was significantly higher in HFHI than in HF group; within the lesion, macrophages with intense iron staining were observed. Hepatic expression of TNFα was higher in HFHI than that in HF group. There was no significant change in hepatic oxidative stress, gut microbiota or serum endotoxin levels between HF and HFHI groups. These results suggested that dietary iron supplementation enhances experimental steatohepatitis induced by long-term high-fat diet feeding in rats. Iron-laden macrophages can play an important role in the enhancement of hepatic inflammation.