Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload, and the cellular processes which result in the observed pathological changes. In addition, the variability in outcome and the compensatory response which likely modulates the effect of increased iron levels are not understood. In order to provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with mass spectrometry (MALDI/MS) we studied hepatic iron overload induced by carbonyl iron-rich diet in mice, and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely urea cycle, fatty acid oxidation and the methylation cycle. This pattern of changes likely reflects compensatory and pathologic changes associated with liver iron overload and provides a window into these processes.