H3 K27–altered spinal cord diffuse midline glioma (H3-SCDMG) poses therapeutic challenges. Analysis of 73 clinical samples revealed heightened proliferation in H3-SCDMG versus wild-type tumors, suggesting therapeutic vulnerabilities. Drug screening identified cytarabine (Ara-C) as highly effective in inhibiting proliferation in H3 K27M cell models, recently established patient-derived cells, and patient-derived xenograft models. Mechanistically, Ara-C can suppress tumor growth through DNA damage, cell-cycle arrest, and apoptosis. An investigator-initiated clinical trial involving four patients showed benefits in three cases. In addition, a subset of cells exhibited senescence and senescence-associated secretory phenotype post–Ara-C treatment, accompanied by several immune checkpoint ligands’ up-regulation and more immune cell infiltration. Combining Ara-C with dual Programmed cell death protein 1 (PD-1) and TIGIT blockade emerged as a promising strategy to disrupt immune evasion by senescent cells, enhancing antitumor responses. These findings highlight Ara-C’s potential as a monotherapy and in synergy with immunotherapy for H3-SCDMG, offering potential strategies for clinical management.