Studying cell differentiation and transformation allows a better understanding of the mechanisms involved in the initiation and the evolution of cancer. The role of proteins which participate in these processes is dependent on their location within the cell. Determining the subcellular localization of proteins or the changes in localization is, therefore, paramount in elucidating their role. Using quantitative mass spectrometry, we characterized the protein expression and subcellular localization of nearly 5000 proteins from seven different colorectal cancer (CRC) cell lines, as well as normal colon fibroblasts and intestinal epithelial cells. This cellular characterization allowed the identification of colon cancer-associated proteins with differential expression patterns as well as deregulated protein networks and pathways. Indeed, our results demonstrate differential expression of proteins involved in cell adhesion, cytoskeleton, and transcription in colon cancer cells compared to normal colon-derived cells. Pathway analyses identified different cellular functions, including endocytosis and eIF2 signaling, whose deregulation correlates with mutations found in the different CRC phenotypes. Our results provide an unbiased, quantitative and high-throughput approach to measure changes in protein expression and subcellular protein locations in different CRC cell lines.