DJ-1 is frequently overexpressed in a large variety of solid tumors, but the DJ-1 expression in laryngeal squamous cell cancer and its clinical/prognostic significance is unclear. We aimed to evaluate DJ-1 protein expression in glottic squamous cell carcinoma (GSCC) and to correlate this with clinicopathological data including patient survival. The expression of DJ-1 in GSCCs (60) and adjacent normal tissue (44) was assessed by immunohistochemistry and western blot analysis. In addition, the role of DJ-1 was investigated in tumorigenesis by transfecting DJ1-specific siRNA into laryngeal squamous cell carcinoma (LSCC) Hep-2 cells. Our data showed that positive expression of DJ-1 was found in 85% of GSCCs. In univariate survival analysis of the GSCC cohorts, a highly significant association between DJ-1 expression with shortened patient overall survival (5-year survival rate 92.9%vs 66.6%; P = 0.001; log rank test) was demonstrated. In multivariate analyses, DJ-1, tumor grading, and pT status were significant prognostic parameters for shortened patient overall survival. Furthermore, siRNA targeting DJ-1 can effectively inhibit DJ-1 expression, resulting in enhanced apoptosis and less proliferation of Hep-2 cells. We concluded that DJ-1 overexpression might be a novel independent molecular marker for poor prognosis (shortened overall survival) of patients with GSCC.