Glioblastoma stem cell (GSC) is the major cause of glioblastoma multiforme (GBM) chemotherapy failure. Hypoxia is one of the determinants of GSC. NFκB plays a pivotal link between hypoxia and cancer stem cells (CSCs). Disulfiram (DS), an antialcoholism drug, has very strong NFκB-inhibiting and anti-CSC activity. In this study, the in vitro anti-GSC activity of DS and in vivo anti-GBM efficacy of poly lactic-co-glycolic acid nanoparticle-encapsulated DS (DS-PLGA) were examined. We attempt to elucidate the molecular network between hypoxia and GSCs, and also examined the anti-GSC activity of DS in vitro and in vivo. The influence of GSCs and hypoxia on GBM chemoresistance and invasiveness was studied in hypoxic and spheroid cultures. The molecular regulatory roles of NFκB, HIF1α and HIF2α were investigated using stably transfected U373MG cell lines. The hypoxia in neurospheres determines the cancer stem cell characters of the sphere-cultured GBM cell lines (U87MG, U251MG, U373MG). NFκB is located at a higher hierarchical position than HIF1α/HIF2α in hypoxic regulatory network and plays a key role in hypoxia-induced GSC characters. DS inhibits NFκB activity and targets hypoxia-induced GSCs. It showed selective toxicity to GBM cells, eradicates GSC and blocks migration and invasion at very low concentrations. DS-PLGA efficaciously inhibits orthotopic and subcutaneous U87MG xenograft in mouse models with no toxicity to vital organs.