Iron-mediated organ damage is common in patients with iron overload diseases, namely hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days and protein expression changes initiated by the iron overload were studied by 2D-electrophoresis and mass spectrometry. From a total of 1060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them, eleven identified proteins were up-regulated whereas eight showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.