The global change in protein abundance in colorectal cancer (CRC) and its contribution to tumorigenesis have not been comprehensively analyzed. In this study, we conducted a comprehensive proteomic analysis of paired tumors and adjacent tissues (AT) using high-resolution Fourier-transform mass spectrometry and a novel algorithm of quantitative pathway analysis. 12380 proteins were identified and 740 proteins that presented a 4-fold change were considered a CRC proteomic signature. A significant pattern of changes in protein abundance was uncovered which consisted of an imbalance in protein abundance of inhibitory and activating regulators in key signal pathways, a significant elevation of proteins in chromatin modification, gene expression and DNA replication and damage repair, and a decreased expression of proteins responsible for core extracellular matrix architectures. Specifically, based on the relative abundance, we identified a panel of 11 proteins to distinguish CRC from AT. The protein that showed the greatest degree of overexpression in CRC compared to AT was Dipeptidase 1 (DPEP1). Knockdown of DPEP1 in SW480 and HCT116 cells significantly increased cell apoptosis and attenuated cell proliferation and invasion. Together, our results show one of largest dataset in CRC proteomic research and provide a molecular link from genomic abnormalities to the tumor phenotype.