Colorectal cancer is one of the most frequent disease in both genders, and is mostly sporadic, associated with a variety of environmental risk factors. The gastrointestinal tract, especially the colon, is constantly exposed to presence of reactive oxygen (ROS) and nitrogen (RNS) species derived from different compounds, like oxidized food debris and toxins. Identification of proteins modified by specifically oxidative/nitrosative injury in affected tissues allows determination of proteins more prone to inactivation by ROS/RNS, which is a fundamental step in linking well-established pathological hallmarks to oxidative/nitrosative changes. We have identified oxidatively damaged (carbonylated) proteins in different stages of colorectal cancer (Dukes A, B and C) compared to non-tumor tissue of the same patient with two dimensional differential gel-electrophoresis and mass spectrometry. In total, 102 carbonylated proteins were identified. There were no changes in protein carbonylation related only to stage A, 16 carbonylated proteins were related only to stage B and 46 proteins related only to stage C. Seven carbonylated proteins were mutual to stages A, B and C, 33 mutual proteins to stages B and C, while there were no mutual carbonylated proteins related to stages A and B, and stages A and C. In total, tissues of 18 patients were analysed, six patients per each stage. The level of malondialdehyde, secondary products during lipid peroxidation and oxidative agens for protein carbonylation generation, was also determined. Our study showed there are significant differences in protein and lipid oxidative damage that differs between tumor and non-tumor tissue in different stages of colorectal cancer and is increasing through the cancer stages. Carbonylated proteins identified in this study belong to cancer-related signaling pathways, antioxidative enzymatic system, metabolic pathways (oxidative phosphorylation the most), and might be potential new biomarkers of colorectal cancer. If validated on other larger number of samples and different tumor types, this finding that differences in emergence of the malignant phenotypes can be a direct functional consequence of oxidative damage to relevant proteins, would make large contribution to better understanding of the ethiology of colorectal cancer and could be the starting point for novel preventive and therapeutic strategies.