The focus of this study was to use differential protein expression to investigate operative pathways in early stages of human colon cancer. Colorectal cancer represents an ideal model system to study the development and progression of human tumors, and the proteomic approach avoids overlooking posttranslational modifications not detected by microarray analyses and the limited correlation between transcript and protein levels. Colon cancer samples, confined to the intestinal wall, were analyzed by expression proteomics and compared with matched samples from normal colon tissue. Samples were processed by two-dimensional gel electrophoresis, and spots differentially expressed and consistent across all patients were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses and by Western blot analyses. After differentially expressed proteins and their metabolic pathways were analyzed, the following main conclusions were achieved for tumor tissue: 1) a shift from beta-oxidation, as the main source of energy, to anaerobic glycolysis was observed owed to the alteration of nuclear- versus mitochondrial-encoded proteins and other proteins related to fatty acid and carbohydrate metabolism; 2) lower capacity for Na(+) and K(+) cycling; and 3) operativity of the apoptosis pathway, especially the mitochondrial one. This study of the human colon cancer proteome represents a step toward a better understanding of the metabolomics of colon cancer at early stages confined to the intestinal wall.