Background: In our previous study, it was observed that Serine Protease Inhibitor Kazal-type 5 (SPINK5) was down-regulated in head and neck squamous cell carcinoma (HNSCC) using oligonucleotide microarrays.However, the function of SPINK5 in the pathological of HNSCC and its clinical significance remain obscure.Methods: In this study, the SPINK5 mRNA and protein level were measured in 9 HNSCC cell lines and in clinical specimens by means of reverse transcription-polymerase chain reaction (RT-PCR), real time PCR and western blotting.Immunohistochemistry was used to measure SPINK5 protein level in tissue microarray contains 101 HNSCC samples.The biological function of SPINK5 on cancerous cell was investigated using transfection technique in vitro and xenografted tumor in vivo.Kaplan Meier survival analysis was used to determine the correlation of SPINK5 expression with overall survival probability.The univariate and multivariate Cox proportional hazards regression model was used to evaluate various parameters associated with clinical outcome.Results: Down-regulation of SPINK5 was found in two thirds HNSCC cell lines and in 85.7% HNSCC specimens(P < 0.0001).We found that reducing SPINK5 expression in WU-HN6 cells lead to the cell proliferation, plate colony and cell invasion were significantly increased, while exogenous expression of SPINK5 in WU-HN13 and HN30 cells, the cell proliferation, plate colony and invasion of cells were remarkably reduced with increased of G1 cell cycle (P=0.0001, P=0.003, respectively).Kaplan Meier survival analysis revealed that patients with HNSCC who had lower SPINK5 expression had significantly inferior overall survival compared to those who expressed higher SPINK5 (P=0.0017).Univariate and multivariate Cox proportional hazards regression model analysis revealed that in addition to the lymph nodes and TNM stage, SPINK5 expression could also used an independent predictor of prognosis for patients with HNSCC (HR=0.114, 95% C1∶0.044-0.292, P < 0.001).Conclusions: Our results demonstrate that SPINK5 down-regulated in HNSCC, this gene play the role of tumor suppressor gene in the carcinogenesis of HNSCC.SPINK5 protein expression can be serving as an independent prognostic predictor for patients with HNSCC.