Glioblastoma is a high-grade glioma with a median survival time of 14-16 months. While glioblastoma has been shown to exhibit a distinct lipidomic profile compared to normal astrocytes, the immunological implications of these lipid alterations remain poorly understood. Here, we investigated the antigenic and immunomodulatory properties of glioblastoma-enriched lipids in the context of human iNKT cells. Using LC-MS-based lipidomic analysis, we compared human glioblastoma cell lines to low-grade glioma cell lines and normal human astrocytes and identified an enrichment of sulfatide species and their biosynthetic precursors, b-GalCer. Functional assays revealed that these sulfatides, but not their precursors, activated a human iNKT cell line, as measured by GM-CSF production. Additionally, sulfatides induced CD69 expression in primary iNKT cells ex vivo from both healthy donors and glioblastoma patients. The glioblastoma cell lines with high sulfatide content exhibited elevated expression of UGT8 and GAL3ST1, enzymes essential for sulfatide biosynthesis. Disruption of GAL3ST1 using genetic or pharmacological approaches significantly impaired the ability of the glioblastoma cell lines to activate human iNKT cells, confirming the functional relevance of sulfatide production. Notably, sulfatide-induced iNKT cell activation did not elicit detectable cytokine production in whole PBMC cultures from eight healthy donors, suggesting that they do not provide strong enough stimuli via a TCR. We also found that lyso-sulfatides were highly enriched in glioblastoma. Importantly, lyso-sulfatides suppressed human iNKT cell activation, including α-GalCer-induced responses. This suppression was seen even when APCs were separately pulsed with α-GalCer and sulfatide prior to mixing with iNKT cells. This suggests that the suppression of iNKT cell activation by lyso-sulfatide is not due to competition for CD1d binding. Collectively, these results indicate that glioblastoma-enriched sulfatides modulate iNKT cell responses and may contribute to the immune suppression associated with glioblastoma. Our findings strongly support further investigations of sulfatide metabolism as a potential immunotherapeutic intervention in glioblastoma.