A mismatch between metabolic demand and oxygen delivery leads to microenvironmental changes in solid tumors. The resulting tumor hypoxia is associated with malignant progression, therapy resistance and poor prognosis. However, the molecular mechanisms underlying therapy resistance in hypoxic tumors are not fully understood. The hypoxia-inducible factor (HIF) is a master transcriptional activator of oxygen-regulated gene expression. Transformed mouse embryonic fibroblasts (MEFs) derived from HIF-1alpha-deficient mice are a popular model to study HIF function in tumor progression. We previously found increased chemotherapy and irradiation susceptibility in the absence of HIF-1alpha. Here, we show by single-cell electrophoresis, histone 2AX phosphorylation and nuclear foci formation of gammaH2AX and 53BP1, that the number of DNA double-strand breaks (DSB) is increased in untreated and etoposide-treated HIF-deficient MEFs. In etoposide-treated cells, cell cycle control and p53-dependent gene expression were not affected by the absence of HIF-1alpha. Using a candidate gene approach to screen 17 genes involved in DNA repair, messenger RNA (mRNA) and protein of three members of the DNA-dependent protein kinase complex were found to be decreased in HIF-deficient MEFs. Of note, residual HIF-1alpha protein in cancer cells with a partial HIF-1alpha mRNA knockdown was sufficient to confer chemoresistance. In summary, these data establish a novel molecular link between HIF and DNA DSB repair. We suggest that selection of early, non-hypoxic tumor cells expressing low levels of HIF-1alpha might contribute to HIF-dependent tumor therapy resistance.