Abstract <p>Glioblastoma (GB), the most aggressive brain tumor invariably recurs despite conventional treatment (surgery, chemotherapy with temozolomide (TMZ), radiotherapy), a phenomenon linked to glioma stem cells (GSC). This study demonstrates that inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) sustains the GSC niche and contributes to TMZ resistance<i>. </i>In silico analysis of 695 glioma samples from The Cancer Genome Atlas showed that higher iNOS expression correlates with poor patient survival. In vitro, GSC-enriched cultures (spheres) from GB cell lines (LN229, U87, U251, GL26) exhibited elevated pluripotency (Nestin gene expression), iNOS expression and NO levels threefold higher than more differentiated cell cultures (2D and 3D-spheroids). The iNOS inhibitor S-methylisothiourea (SMT) effectively reduced NO production. Sequential treatment with TMZ followed by TMZ + SMT more effectively reduced viability in 2D and 3D-spheroids growth compared to TMZ alone. In GSC enriched spheres, SMT decreased GSC frequency and growth, and combined TMZ + SMT showed enhanced reduction. Primary human glioma cultures (n = 11) expressed iNOS and their GSC spheres formation efficiency was reduced by SMT. These findings suggest that iNOS/NO axis is critical for GSC maintenance and TMZ resistance. Sequential TMZ followed by iNOS inhibition shows promise as a therapeutic strategy against GB recurrence by targeting GSCs.</p> Key messages <p><UnorderedList Mark="Bullet"> <ItemContent> <p>Elevated iNOS expression correlates with poor glioma prognosis markers.</p> </ItemContent> <ItemContent> <p>Temozolomide acts on more differentiated glioma cells and enriches stem cells.</p> </ItemContent> <ItemContent> <p>iNOS inhibition reduces the stem cell niche, enhancing TMZ sensitization.</p> </ItemContent> </UnorderedList></p>