Differential protein expression was analyzed in carcinoma tissue to determine the correlation between protein levels and the clinical and pathological parameters of patients with colorectal carcinomas (CRC). Two-dimensional electrophoresis (2-DE) revealed 40 protein spots that were differentially expressed at two or greater fold difference in CRC that were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS). Among these proteins, prohibitin (PHB) was found to be overexpressed in CRC. It was selected for Western blot and immunohistochemistry assay in subsequent tissue microassays (TMA). Thirty-five distinct proteins were differentially expressed at least 2-fold among normal and CRC tissues. The expression of 17 proteins, including PHB, was increased by >2-fold in CRC tissues (p<0.01). Immunohistochemistry and Western blot assays found that PHB was overexpressed in CRC cases, and the expression was higher than adenoma and normal tissues (p<0.01), whereas there was no significant difference in expression of PHB between adenoma and normal tissues. Immunohistochemistry also suggested a link between PHB expression and poor differentiation (p<0.01). However, there was no difference in UICC stage, or location of CRC. Survival analysis suggested no significant correlation between PHB expression and poor prognosis. In summary, the overexpression of PHB might be associated with the transformation process from adenoma to CRC. Further, it is a potential diagnostic and differentiation biomarker of CRC in the clinic.