Purpose: The resistance of HNSCC to radiotherapy is partly explained by the presence of Cancer Stem Cells (CSCs) located in hypoxic niches. The protein HIF-1α (Hypoxia-Inducible Factor 1α) is considered as the major transcriptional regulator of the cellular response to oxygen homeostasis. Compared to X-rays, carbon ion efficiency relies on better ballistic properties and a higher relative biological effectiveness independent of the oxygen concentration. This work aims at clarifying the role of HIF-1alpha in the response of CSCs to photon and carbon ion irradiations. Methods: HNSCC-CSCs were transfected with a non-relevant or a siRNA targeting HIF-1α. Oxygen Enhancement Ratio (OER) was calculated at the ratio of dose inducing 10% survival after photon or carbon ion irradiation in hypoxic (1% O2) versus normoxic conditions. DNA Double Strand Breaks (DSBs) were quantified by the γ-H2Ax assay and ROS with a CM-H2DCFDA dye. Results: For two HNSCC cell lines and their CSC subpopulation, in response to photons under hypoxia, an OER>1.2 was associated with HIF-1α expression. This stabilization appears earlier in CSCs than in non CSCs and is correlated with the variation of ROS levels, confirming the adaptive properties of CSCs to hypoxia. Compared with photons, the oxygen effect is canceled after carbon ion exposure (OER=1) and no stabilization of HIF-1α was observed in normoxia. Inhibition of HIF-1α with a siRNA leads to the decrease of CSC survival after both radiations in hypoxic conditions (OER<1). Furthermore, radiosensitization is associated with a significant increase of residual DSBs. Conclusion: These results demonstrate that HIF-1alpha plays a key role in the response of CSCs to photon and carbon ion irradiation. It participates to radioresistance by increasing cell survival and DNA repair and contributes to tumor escape. This makes the HIF-1α targeting an attractive therapeutic challenge. Supported by LabEx PRIMES, FRANCE HADRON, LYRIC and Jean Walter-Zellidja Grant