Glioblastoma is one of the most malignant brain tumors, causing death within two years despite maximal tumor resection and concurrent radio-chemotherapy. Tumor stem cells are thought to be closely related to tumor progression and recurrence and are attractive therapeutic targets. It is common to culture cell lines in serum-free medium with growth factors to stimulate spheres of enriched tumor stem cells. To avoid spontaneous differentiation and cell death in the sphere environment, Pollard et al. formulated an adherent culture method using laminin-coated plates. We here evaluated collagen-1-coated plates, which are superior to laminin-coated plates in handling and cost, as an alternative adherent culture method of CD133 expressing glioblastoma cells. We cultured the human glioblastoma cell line, U87MG, under serum contained medium (SCM) or serum free medium with EGF, FGF2, LIF, B27 and N2 supplements (SFM) in non-coated, laminin or collagen-1-coated plates. The growth morphology in various cultures was evaluated, and the number of living cells in each plate on day 5 after cell seeding was calculated. The RNA expression level of CD133 as a stem cell marker in each plate was examined. Semi-quantitative measurement of CD133 positive cells by immunocytochemistry was performed. In collagen-1-coated plates with SFM, cell lines were cultured in an adherent monolayer. Cell proliferation was statistically encouraged in collagen-1-coated plates. Both laminin-coated plates and collagen-1-coated plates with SFM enhanced the RNA expression of CD133 compared to non-coated plates with SCM. Immunocytochemistry showed a statistically significant increase of CD133 positive cells in collagen-1-coated plates with SFM. Collagen-1-coated plates with SFM was used for cell morphology and cell proliferation of CD133 expression in the U87MG malignant glioma cell line.