Objective: To screen new tumor markers for colorectal cancer (CRC) through analyzing the differential protein expression profiles between CRC and normal mucosa by using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE)-based proteomic techniques. Methods: The differentially expressed proteins were analyzed in six fresh CRC specimens and paired normal mucosa by 2D-DIGE and then identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), respectively. Thereafter, the proteins of interest were verified by immunohistochemical method in 46 cases of CRC and normal mucosal tissues. Results: 2D-DIGE analysis and MALDI-TOF-MS identification showed that the significantly up-regulated proteins in CRC tissues were ubiquinol cytochrome-c reductase core protein 1 (UQCRC1), 14-3-3 ζ and desmin etc., and the significantly down-regulated proteins were carbonic anhydrase (CA) I and CAII etc. (all P<0.05). The results of immunohistochemical staining revealed that the expression intensities of UQCRC1 and 14-3-3 ζ in CRC tissues were 2.43±0.81 and 1.41±1.07 respectively, which were significantly higher than those in normal mucosal tissues (1.80±0.96 and 0.67±0.94) (P<0.001), while the desmin expression had no significant difference between the two tissues (P>0.05); the expression intensities of CA I and CA II in CRC tissues were 1.67±0.52 and 1.18±0.84 respectively, which were significantly lower than those in normal mucosal tissues (2.93±0.25 and 2.80±0.50) (P<0.001). Conclusion: The 2D-DIGE-based proteomic techniques combined with immunohistochemical method are effective means to screen new tumor markers. UQCRC1, 14-3-3 ζ, CA I and CA II may potentially become the novel markers or therapeutic targets of CRC.