ABSTRACTNasopharyngeal carcinoma (NPC) is a common disease in the southern provinces of China with a poor prognosis. To better understand the pathogenesis of NPC and identify proteins involved in NPC carcinogenesis, we applied iTRAQ coupled with two‐dimensional LC‐MS/MS to compare the proteome profiles of NPC tissues and the adjacent non‐tumor tissues. We identified 54 proteins with differential expression in NPC and the adjacent non‐tumor tissues. The differentially expressed proteins were further determined by RT‐PCR and Western blot analysis. In addition, the up‐regulation of HSPB1, NPM1 and NCL were determined by immunohistochemistry using tissue microarray. Functionally, we found that siRNA mediated knockdown of NPM1 inhibited the migration and invasion of human NPC CNE1 cell line. In summary, this is the first study on proteome analysis of NPC tissues using an iTRAQ method, and we identified many new differentially expressed proteins which are potential targets for the diagnosis and therapy of NPC. J. Cell. Biochem. 116: 1431–1441, 2015. © 2015 Wiley Periodicals, Inc.