Glucose transporter GLUT1 is a transmembrane protein responsible for the uptake of glucose into the cells of many tissues through facilitative diffusion. Plasma membrane (PM) localization is essential for glucose uptake by GLUT1. However, the mechanism underlying GLUT1 PM localization remains enigmatic. We find that GLUT1 is palmitoylated at Cys207, and S-palmitoylation is required for maintaining GLUT1 PM localization. Furthermore, we identify DHHC9 as the palmitoyl transferase responsible for this critical posttranslational modification. Knockout of DHHC9 or mutation of GLUT1 Cys207 to serine abrogates palmitoylation and PM distribution of GLUT1, and impairs glycolysis, cell proliferation, and glioblastoma (GBM) tumorigenesis. In addition, DHHC9 expression positively correlates with GLUT1 PM localization in GBM specimens and indicates a poor prognosis in GBM patients. These findings underscore that DHHC9-mediated GLUT1 S-palmitoylation is critical for glucose supply during GBM tumorigenesis.