The present study analyzed the correlation of DNA polymerase β (DNA polβ) promoter mutations and activity in esophageal squamous cell carcinoma (ESCC). The DNA polβ promoter was amplified from 108 ESCC samples and adjacent paracancerous samples by PCR and cloned into the pGL3-enhancer luciferase vector. The recombined vectors were transfected into esophageal carcinoma cells (EC9706, Eca109, and KYSE30), and luciferase activity was detected using dual luciferase reporter gene technology. Eleven polβ promoter mutations were identified and submitted to GenBank. The mutation rate of the DNA polβ promoter was higher in ESCC tissues (36/108, 33.3 %) than in the paired paracancerous tissues (21/108, 19.4 %) (P = 0.021). The C → A mutation at locus -37 was the hotspot mutation in cancerous tissues, and its frequency was higher in ESCC tissues (26/108) than in paracancerous tissues (7/108) (P = 0.00). The highest relative luciferase activity (RLA) was observed in the DNA polβ promoter, with a C → A mutation at -37. Significant differences in RLA were observed between mutant DNA polβ promoters (except for C detected at -19, T → C at -194, C → A at -37, and T → C at 30) and the wild-type DNA polβ promoter (P = 0.000), and RLA was significantly higher in ESCC tissues than in paracancerous tissues (P = 0.003). Our findings suggest that the upregulation of transcriptional activity induced by mutations in the DNA polβ promoter in ESCC tissues may be one of the molecular mechanisms mediating abnormal overexpression of DNA polβ in ESCC.