We previously reported the in vitro and in vivo efficacy of N,N-bis(2-chloroethyl)-2-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)propenamide, a prodrug that targeted the mitochondria of glioblastoma (GBM). The mitochondrial enzyme monoamine oxidase B (MAOB) is highly expressed in GBM and oxidizes an uncharged methyl-tetrahydropyridine (MP-) moiety into the mitochondrially-targeted cationic form, methyl-pyridinium (P+-). Coupling this MAOB-sensitive group to a nitrogen mustard produced a prodrug that damaged GBM mitochondria and killed GBM cells. Unfortunately, the intrinsic reactivity of the nitrogen-mustard group and low solubility of MP-MUS precluded clinical development. In our second generation prodrug, MP-Pt(IV) we coupled the MP-group to an unreactive cisplatin precursor. The enzymatic conversion of MP-Pt(IV) to P+-Pt(IV) was tested using recombinant human MAOA and rhMAOB. The generation of cisplatin from Pt(IV) by ascorbate was studied optically and using mass-spectroscopy. Efficacy toward primary GBM cells and tumors was studied in vitro and in an intracranial patient-derived xenograft mice GBM model. Our studies demonstrate that MP-Pt(IV) is selectively activated by MAOB. MP-Pt(IV) is highly toxic toward GBM cells in vitro. MP-Pt(IV) toxicity against GBM is potentiated by elevating mitochondrial ascorbate and can be arrested by MAOB inhibition. In in vitro studies, sub-lethal MP-Pt(IV) doses elevated mitochondrial MAOB levels in surviving GBM cells. MP-Pt(IV) is a potent chemotherapeutic in intracranial patient-derived xenograft mouse models of primary GBM and potentiates both temozolomide (TMZ) and TMZ-chemoradiation therapies. MP-Pt(IV) was well tolerated and is highly effective against GBM in both in vitro and in vivo models.