Accumulating evidence has demonstrated that the presence of a subset of cells in several types of brain tumor, termed brain cancer stem cells, are responsible for tumor recurrence following chemotherapy or radiotherapy. The isolation and characterization of side population (SP) cells in several types of solid tumor using Hoechst dye has become a powerful technique for obtaining cancer stem cells (CSCs). In the present study, cancer stem‑like SP cells were isolated from the human glioblastoma cell line MG‑12 using the Hoechst 33342 dye exclusion technique. Flow cytometric analysis revealed that the cell line MG‑12 contained 3.2% SP cells, which was reduced to 0.5% in the presence of verapamil, an inhibitor of ATPase‑binding cassette (ABC) transporters. Reverse‑transcription quantitative polymerase chain reaction analysis revealed that the isolated SP cells exhibited increased expression of stem cell markers, including Nestin, Notch 1, octamer‑binding transcription factor 4 (Oct‑4), epithelial cell adhesion molecule (EpCAM) and also ABC transporter protein ABCG2. Additionally, using western blot analysis it was demonstrated that SP cells exhibit positivity and have a higher expression of CD133, CD44, EpCAM, Oct‑4 and B‑cell lymphoma 2. Furthermore, it was demonstrated that the isolated SP cells undergo rapid proliferation, have a high propensity to form tumor spheres and also have a high survival rate following treatment with 5‑fluorouracil. Therefore, the present findings suggest that SP cells of the glioblastoma MG‑12 cell line share characteristics of CSCs. Therefore, the increased expression of stem cell markers and ABCG2 protein may interact with each other and be responsible for drug and apoptotic resistance, tumor recurrence and invasion.