Tumor hypoxia has been reported to cause a functional loss in DNA mismatch repair (MMR) system as a result of down-regulation of MMR genes, although the precise molecular mechanisms remain unclear. In this study, we focused on the down-regulation of a key MMR gene, MLH1, and demonstrated that hypoxia-inducible transcription repressors, DEC1 and DEC2, participated in its transcriptional regulation via their bindings to E-box-like motif(s) in MLH1 promoter region. In all cancer cell lines examined, hypoxia increased expression of DEC1 and DEC2, known as hypoxia-inducible genes, but decreased MLH1 expression in an exposure time-dependent manner at both the mRNA and protein levels. Co-transfection reporter assay revealed that DEC1 and, to greater extent, DEC2 as well as hypoxia repressed MLH1 promoter activity. We further found that the action was remarkably inhibited by trichostatin A, and identified a possible DEC-response element in the MLH1 promoter. In vitro electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that DEC1 or DEC2 directly bounds to the suggested element, and transient transfection assay revealed that overexpression of DEC2 repressed endogenous MLH1 expression in the cells. Hypoxia-induced DEC may impair MMR function through repression of MLH1 expression, possibly via the histone deacethylase (HDAC)-mediated mechanism in cancer cells.