Simple SummaryHypoxia-Inducible Factor 1α (HIF-1α), the main regulator of the oxygen homeostasis, promotes cancer cell survival through proliferation, angiogenesis, metastasis and radioresistance. Previously, our group demonstrated that silencing HIF-1α under hypoxia leads to a substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and carbon-ions, probably resulting from an accumulation of deleterious complex DNA damage. In this study, we aimed at determining the potential role of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, under hypoxia, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). Silencing HIF-1α under hypoxia led us to demonstrate the involvement of this transcriptional regulator in DSB repair in non-CSCS and CSC, thus highlighting its targeting together with radiation as a promising therapeutic strategy against radioresistant tumor cells in hypoxic niches.Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.