SummaryBackground : Preliminary clinical and experimental results suggest that iron can modify hepatocytes’ insulin sensitivity by interfering with insulin receptor and intracellular insulin signalling.Aim : To evaluate in vivo the influence of iron on insulin resistance and insulin release in patients with non‐alcoholic fatty liver disease and in vitro the interaction between iron and insulin sensitivity by analysing the effect of iron manipulation on insulin receptor expression in hepatoblastoma HepG2 cell line.Results : Insulin resistance evaluated by homeostatis model assessment (HOMA)‐insulin resistance significantly decreased after diet, and a further reduction was observed after phlebotomies. Iron depletion by desferrioxamine increased by twofold the 125I‐insulin‐specific binding, whereas iron addition reduced insulin binding, similarly to cells exposed to high glucose concentration.Conclusion : Iron status affects insulin sensitivity by modulating the transcription and membrane expression/affinity of insulin receptor expression in hepatocytes and influencing insulin‐dependent gene expression suggesting that increased insulin clearance and decreased insulin resistance may contribute to the positive effect of iron depletion in patients with non‐alcoholic fatty liver disease.