Hydrogen sulfide (H₂S) functions as a signaling molecule and a cytoprotectant. The pathway to produce H₂S includes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). 3MST produces H₂S from 3-mercaptopyruvate that is provided from l-cysteine and α-ketoglutarate (α-KG) by cysteine aminotransferase (CAT) and from d-cysteine by d-amino acid oxidase (DAO). Recently, a new concept emerges in H₂S biology, showing tumor cells upregulate the production of H₂S and utilize it to promote tumor growth and chemotherapy resistance. In this study, we investigated the production of H₂S in cell lines derived from glioblastoma, the most common- and aggressive-type of brain tumors among adults. Lysates of glioblastoma cells produced H₂S from l-cysteine and α-KG. In the absence of α-KG, the production of H₂S was significantly decreased, suggesting that H₂S production is dependent on the activity of CAT. The production of H₂S from l-cysteine and α-KG was correlated with the amount of bound sulfane sulfur, a storage form of H₂S. Unlike the l-cysteine pathway, d-cysteine pathway does not operate in the glioblastoma cells. Further investigation will be needed to clarify whether regulation of the l-cysteine pathway changes proliferation and anti-cancer drug resistance of the glioblastoma cells.