Two-dimensional (2D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry was utilized to compare the protein expression profiles between two colorectal cancer (CRC) cell lines with high and low metastatic potentials, SW620 and SW480. 10 metastasis-associated proteins were identified and further validated by Western blotting and/or semi-quantitative RT-PCR analysis. Of the identified proteins, the expressions of phosphoglycerate mutase 1, phosphatidylethanolamine binding protein and high-mobility group box 1 were elevated in SW620 cells. However, heat shock protein 27, annexin Ⅰ, methylthioadenosine phosphorylase, cofilin-1 and epidermal fatty acid binding protein were down-regulated in SW620 cells. Most of the candidate proteins have been evident to be somehow associated with various aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity. These results provide the basis for searching for potential markers for CRC prognosis and give some clues to elucidate the mechanism of CRC metastasis.