The ability of a promoter to initiate transcription is important for the control of gene expression. Mutations in the DNA polymerase beta (po1β) promoter may affect the transcription of this gene; however, the relationship between these mutations and the upregulation of the expression of po1β remains unclear. Therefore, in the present study, three po1β promoter mutants (M1, -37 C→A; M2, -114 G→A, -37 C→A; M3, -194 T→C) were generated to examine the effect of promoter mutations on polβ gene expression and sensitivity to cisplatin. We found that the M1 and M2 mutant polβ promoter constructs showed higher RLA than the wild-type polβ promoter (P < 0.01), whereas the activity of the M3 polβ promoter did not differ significantly from that of the wild-type polβ promoter (P > 0.05). The expression levels of polβ mRNA and protein were significantly higher (P < 0.01) and the sensitivity to cisplatin was significantly lower (P < 0.05) in Eca9706(-/-)-M1 and Eca9706(-/-)-M2 cells than in Eca9706(-/-)-W. The expression levels of polβ mRNA and protein and the sensitivity to cisplatin were not significantly different between Eca9706(-/-)-M3 and Eca9706(-/-)-W cells (P > 0.05).These results revealed that specific mutations of the polymerase beta gene promoter significantly enhanced the gene's transcriptional activity. These mutations correspondingly increased the gene's mRNA and protein product, at the same time reduced the esophageal cancer cells' sensitivity to cisplatin.