Oleandrin is a glycoside that inhibits the ubiquitous enzyme Na⁺/K⁺-ATPase. In addition to its known effects on cardiac muscle, recentin vitroandin vivoevidence highlighted its potential for anticancer properties. Here, we evaluated for the first time the effect of oleandrin on brain tumors. To this aim, mice were transplanted with human or murine glioma and analyzed for tumor progression upon oleandrin treatment. In both systems, oleandrin impaired glioma development, reduced tumor size, and inhibited cell proliferation. We demonstrated that oleandrin does the following: (1) enhances the brain-derived neurotrophic factor (BDNF) level in the brain; (2) reduces both microglia/macrophage infiltration and CD68 immunoreactivity in the tumor mass; (3) decreases astrogliosis in peritumoral area; and (4) reduces glioma cell infiltration in healthy parenchyma. In BDNF-deficient mice (bdnftm1Jae/J) and in glioma cells silenced for TrkB receptor expression, oleandrin was not effective, indicating a crucial role for BDNF in oleandrin's protective and antitumor functions. In addition, we found that oleandrin increases survival of temozolomide-treated mice. These results encourage the development of oleandrin as possible coadjuvant agent in clinical trials of glioma treatment.SIGNIFICANCE STATEMENTIn this work, we paved the road for a new therapeutic approach for the treatment of brain tumors, demonstrating the potential of using the cardioactive glycoside oleandrin as a coadjuvant drug to standard chemotherapeutics such as temozolomide. In murine models of glioma, we demonstrated that oleandrin significantly increased mouse survival and reduced tumor growth both directly on tumor cells and indirectly by promoting an antitumor brain microenvironment with a key protective role played by the neurotrophin brain-derived neurotrophic factor.