Glioblastoma is the most frequent primary malignant brain tumor in adults. Oxysterols are oxidation products of cholesterol generated by enzymatic reactions. 27-hydroxycholesterol (27-HC), an oxysterol, is an abundant metabolite of cholesterol. 27-HC significantly accelerates mammary cancer growth, proliferation and progression in experimental models. However, to the best of our knowledge, the effect of 27-HC on glioblastoma has not been studied. Therefore, the present study aimed to determine the exact role of 27-HC in glioblastoma. The present study demonstrated that 27-HC promoted proliferation, epithelial to mesenchymal transition, colony formation, migration and invasion of U251 and U118 MG glioblastoma cells. Treatment with 27-HC was also associated with an increase in the formation of glioblastoma-initiating cells in U251 and U118 MG cell lines. Additionally, it was observed that high levels of 27-HC in glioblastoma tissues were associated with poor outcome in patients. In conclusion, 27-HC, a primary metabolite of cholesterol, may serve an important role in the progression of glioblastoma.