AbstractHead and neck squamous cell carcinoma (HNSCC) represents a major health concern worldwide. We applied the matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) to analyze paired normal (N) and tumor (T) samples from head and neck squamous cell carcinoma as well as liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis in HNSCC cell lines to identify tumor-associated biomarkers. Our results showed a number of proteins found to be over-expressed in HNSCC. We identified thymosin beta-4 X-linked (TMSB4X) is one of the most significant candidate biomarkers. Higher TMSB4X expression in the tumor was found by N/T-paired HNSCC samples at both RNA and protein level. Overexpression of TMSB4X was found significantly associated with poor prognosis of overall survival (OS, P = 0.006) and recurrence-free survival (RFS, P = 0.013) in HNSCC patients. Silencing of TMSB4X expression in HNSCC cell line reduced the proliferation and invasion ability in vitro, as well as inhibited the cervical lymph node metastasis in vivo. Altogether, our global proteomics analysis identified that TMSB4X is a newly discovered biomarker in HNSCC whose functions resulted in enhanced proliferation and metastasis in vitro and in vivo. TMSB4X may be a potential therapeutic target for treating HNSCC patients.