Colorectal cancer is still a major health burden worldwide, and its diagnosis has not improved in recent years due to a lack of appropriate diagnostic serum markers. Aiming to find new diagnostic proteins, we applied the proteomic DIGE technology to analyze changes in the secretome before/after differentiation of the colon adenocarcinoma Caco-2 cell line, an accepted in vitro model to study colorectal tumorigenesis. When the secretomes from undifferentiated (tumor-like) and differentiated cells (resembling healthy enterocytes) were compared, we found 96 spots differentially expressed. After MS/MS analysis, 22 spots corresponding to 15 different proteins were identified. Principal component analysis demonstrated these 22 spots could serve as a discriminatory panel between the tumor-like and normal-like cells. Among the identified proteins, the translationally-controlled tumor protein (TCTP), the transforming growth factor-beta-induced protein ig-h3 (TGF beta Ip), and the Niemann-Pick disease type C2 protein (NPC2) are interesting candidates for future studies focused on their utility as serum biomarkers of colorectal cancer.