Objective: The aim of the study was to screen differentially expressed proteins between left- and right-sided colon cancers by proteomics techniques and provide molecular genetic basis for oncobiological difference between left- and right-sided colon cancers. Methods: Tissue samples including left- and right-sided colon cancers were collected and preserved in the –80 ℃refrigeratory. In the first part of our experiment, protein separating was performed by using two-dimensional gel electrophoresis (2-DE) and the images of the gels were acquired by the scanner and then analyzed to find the differentially expression protein-spots in different groups. The peptide mass fingerprintings (PMF) was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the proteins were identified by data searching in the Mascot-database. Differentially expression proteins were assayed by RT-PCR, Western blot, and immunohistochemical methods. Results: The 55 differentially expressed protein spots were screened and 23 spots of them were identified. Compared to right-sided colon cancer, 15 proteins up-regulated and 8 proteins down-regulated including HSP27 in left-sided colon cancer. HSP27 expressed higher in right-sided than in left-sided colon cancers by RT-PCR, Western blot and immunohistochemical methods. Conclusion: There were differentially expressed proteins between left- and right-sided colon cancers, especially differences in HSP27 expression in mRNA and protein level, which were molecular genetic basis for oncobiological difference between left- and right-sided colon cancers.