Glioblastoma multiforme (GBM), known as astrocytoma grade IV, is the most common primary malignant brain tumor and one of the most lethal forms of cancer with a median survival rate of approximately 15 months after standard treatment. Currently, the chance of increasing the survival rate is found in therapy with doxorubicin (DOX) delivered to the central nervous system by polymeric carriers. In this study, we synthesized and characterized by NMR spectroscopy, a vehicle for DOX based on glucoheptoamidated poly (amidoamine) dendrimers of third generation (G3gh) and its derivative substituted with 4 residues of DOX attached via succinate linkers (G3gh4DOX). The pH dependent DOX release profile from the well soluble G3gh4DOX indicates a slow release that is significantly higher in an acidic environment due to hydrolytic cleavage of an amide bond between DOX and the succinate linker, not the ester bond between succinate linker and the glucoheptoamide substituent. Biological studies demonstrate efficient grade IV human U-118 MG glioma cells damage by G3gh4DOX conjugates at very low concentrations, with IC50 < 1 μM after 24–72 h of incubation. The conjugate studied was about 2.7–4.5 fold more toxic than DOX alone. G3gh4DOX killed glioma cells through a more desirable apoptosis pathway and inhibited their proliferation with cell cycle-arrest at the G2/M phase. The effect of G3gh4DOX was probably the result of action of the entire conjugate, but not DOX alone. Therefore, G3gh4DOX seems to be a promising candidate for local glioma therapy.