Glioma stem cells (GSCs) are a minority population of glioma cells that regarded as the cause of tumor formation and recurrence. Identifying new molecular strategies targeting GSCs must be urgently developed to treat glioblastoma. In this study, one of CD98 light chain-L type amino acid transporter 1 (LAT1) was found as a potential GSC marker. LAT1 served as EAA transporter has been shown to be closely related with tumor invasion, metastasis, angiogenesis, and radiosensitivity.LAT1+ and LAT1- glioma cells were sorted by flow cytometry. Cellular immunofluorescence, sphere-formation arrays, and in vitro limiting dilution experiments were used to identify cell stemness. Differentiated glioma stem cells were cultured, and the expressions of β-tubulinIII, GFAP, and LAT1 were detected by Western blot. Nude mouse models were constructed to observe tumor formation and metastasis in nude mice.LAT1+ glioma cells were testified a small percentage of all cells and selected as the subsequent sorting marker. LAT1+ cells were separated from U87 and U251 cells could express high level of stem cell markers, and possessed GSC properties including self-renewal ability and multi-directional differentiation potential. But LAT1- cells did not have these characteristics. In addition, LAT1+ cells were able to generate tumors in vivo, tumor size of LAT1+ cells formed were much bigger than that of LAT1- cells.Our study, including molecular, cell, vitro and vivo experiments, has shown that LAT1+ cells possess GSC properties, and present for the first time that LAT1 can be used as a new marker for GSCs screening.Copyright © 2022 Elsevier B.V. All rights reserved.