To better understand the mechanism underlying colorectal carcinoma (CRC) genesis or metastasis, and to search for potential markers for CRC prognosis, a comparative proteomic analysis was performed on CRC tissue. Proteins were extracted from normal colorectal mucosa, non‐metastatic CRC (nmCRC) and metastatic CRC (mCRC) tissue samples. Protein profiling of each sample was performed by two‐dimensional electrophoresis coupled with MALDI‐TOF MS, followed by confirmation by Western blotting. Thirty‐one proteins were found to be differentially expressed between normal mucosa, nmCRC and mCRC tissue. In 126 paraffin‐embedded CRC samples, three differentially expressed proteins, identified as LASP‐1, S100A9 and RhoGDI by proteomic analysis, were detected by immunohistochemical staining to determine the clinicopathological characteristics of these proteins in CRC. Increased expression levels of these proteins were found in CRC, especially mCRC, compared with normal mucosa. The results provide the basis for searching for potential markers for CRC genesis and metastasis, and also provide clues for elucidating the mechanism of CRC progression. The pattern changes identified have the potential to be used for the design of marker panels for assistance in diagnostic and therapeutic strategies in CRC.