Esophageal cancer ranks sixth in cancer mortality worldwide and patients with esophageal squamous cell carcinoma (ESCC) have a poor prognosis with a 5-year survival rate of less than 10%. Elucidation of the mechanisms of carcinogenesis and tumor progression in esophageal cancer is urgently required to develop targets for therapy and prognostic biomarkers. In the present study, the expression and regulatory mechanism of the differentially expressed in normal and neoplastic cells domain containing 2D (DENND2D), which is a regulator of Rab GTPases, were investigated to explore its potential as a tumor suppressor gene for ESCC. The level of DENND2D mRNA expression in ESCC cell lines and surgical specimens was determined using a quantitative real-time reverse transcription-polymerase chain reaction assay, and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanism of DENND2D expression in ESCC. The level of DENND2D mRNA expression was reduced in 8/9 ESCC cell lines and in 59/65 surgical specimens, and the mean expression levels were significantly lower in cancerous tissues compared to corresponding normal tissues (p<0.001). The expression pattern of DENND2D protein and mRNA was consistent. Downregulation of DENND2D mRNA in ESCC tissues was identified as an independent prognostic factor in multivariate analysis (hazard ratio, 2.194; p=0.039). The DENND2D promoter was methylated in 5/9 ESCC cell lines, and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in ESCC tissues (64.6%) and was significantly associated with downregulation of DENND2D mRNA expression (P=0.008). Taken together, our data suggest that DENND2D is a candidate tumor suppressor gene that was inactivated by promoter hypermethylation in patients with ESCC and may serve as a novel biomarker of ESCC.