Esophageal cancer is one of the most common malignancies worldwide. DNA N6-methyladenine (6mA) has been well-studied in prokaryotes, while the distribution and biological functions of DNA 6mA in eukaryotic cells remain to be elucidated. Recently, DNA 6mA epigenetic modification was found in human gastric and liver cancers. To explore the status of DNA 6mA epigenetic modification in esophageal cancer, 101 cases of human esophageal squamous cell carcinoma (ESCC) and matched adjacent normal tissue samples were analyzed by dot blot assay. The levels of genomic DNA 6mA were significantly higher in ESCC tissue samples than in matched adjacent normal tissue samples (P<0.001). Increased DNA 6mA was associated with poor tumor differentiation (P<0.05), while no association was found between 6mA modification and gender, age, tumor size, TNM stage, lymph node metastasis, smoking, alcohol intake, or family history (all P>0.05). In conclusion, DNA 6mA epigenetic modification increased in human ESCC and may serve as a prognostic marker.