Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stem-like cells (GSCs), by culturing patient-derived primary GSCs as well as a GSC cell line in glucose-restricted, beta-hydroxybutyrate-containing medium (BHB-G(low)) which was used to mimic clinical KD treatment. GSCs cultured in BHB-G(low) medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished self-renewal in vitro, and reduced tumorigenic capacity in vivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHB-G(low) medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHB-G(low) medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxia-inducible factor 1 and B-cell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHB-G(low) medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.