DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNAPKis activated by mild hypoxia conditions (0.1–1% O2) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisationfrom a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed byactivation and recruitment of the XRCC4–DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNAPK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor,leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cellsand treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PKactivation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor(NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and onesubsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation,which positively regulate cellular oxygen-sensing and oxygen-signalling pathways.