Abstract Pediatric midline high-grade gliomas (pmHGG) are aggressive and incurable tumors of the central nervous system. There is a pressing need for novel therapeutic approaches to treat them. Therefore, proactive translational studies wish to go further discovering new targetable proteins and pathways. Our objectives are then to focus on the modulation of microenvironmental extrinsic features like intra-tumor hypoxia. To do so, we looked first on expressions of hypoxia biomarkers in a pool of patient-derived preclinical models of pmHGG and tested oxygen modulations, as well as hypoxia drug targeting. We designed subsequently our work in those models H3.3 mutated to evaluate balance between HIF1 and HIF2 expressions (immunofluorescence, RTqPCR, RNAseq and metabolomics) and to evidence the impact of hypoxia targeting combined to irradiation on cell proliferation, migration and metabolism. Hypoxia is inducing mainly HIF1 expression and its upstream and downstream pathways and is stabilizing HIF2 expression. Both HIFs are part of crucial survival signaling and represent targets to combine with irradiation. The use of their specific inhibitors shows an antiproliferative effect when HIF1 is downregulated. HIF2 inhibitors are stopping HIF2 transcriptional effect letting us uncover new pathways that this hypoxic inducible factor is regulating in pmHGG (stemness, glycolytic and aminoacid metabolism and histone expression). Together with irradiation this anti-hypoxic strategy seems to be highly effective on cell arrest and migration. Those results are confirming central roles of HIFs in pmHGG and their potencies in pmHGG therapies. The therapeutic efficiency is independent from p53 abnormalities in our models.