Hypoxia promotes genetic instability by undefined mechanisms. The transcription factor HIF-1α is crucial for the cellular response to hypoxia and is frequently overexpressed in human cancers, resulting in the activation of genes essential for cell survival. Here, we demonstrate that HIF-1α is responsible for genetic instability at the nucleotide level by inhibiting MSH2 and MSH6, thereby decreasing levels of the MSH2-MSH6 complex, MutSα, which recognizes base mismatches. HIF-1α displaces the transcriptional activator Myc from Sp1 binding to repress MutSα expression in a p53-dependent manner; Sp1 serves as a molecular switch by recruiting HIF-1α to the gene promoter under hypoxia. Furthermore, in human sporadic colon cancers, HIF-1α overexpression is statistically associated with the loss of MSH2 expression, especially when p53 is immunochemically undetectable. These findings indicate that the regulation of DNA repair is an integral part of the hypoxic response, providing molecular insights into the mechanisms underlying hypoxia-induced genetic instability.