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Background Invariant natural killer T (iNKT) cells are CD1d-restricted T cells expressing a semi-invariant T cell receptor (TCR) that recognizes lipid antigens and are involved in the anti-cancer immune response. Studies of a prototypic iNKT cell antigen, α-galactosylceramide (α-GalCer), analogs have shown that ceramide structure affects the activities of mouse iNKT cells. However, much less has been studied on human iNKT cells. This study aimed to investigate the antigenic activity of novel sulfatide-based lipids for human iNKT cells with potential implications for cancer immunotherapy.Methods and Results Eight new sulfatide analogs were synthesized with fluorobenzene at the end of acyl chains and three sphingoid base structures. First, we evaluated the recognition of the sulfatide analogs by the TCR of human iNKT cells by measuring the reactivity of human CD1d-tetramers loaded with sulfatide analogs. Testing peripheral blood mononuclear cells (PBMCs) from three healthy donors showed that one sulfatide analog, which we named SAP-3, was commonly recognized by all donors’ iNKT cells. Next, we examined the activation of ex vivo human iNKT cells derived from PBMCs by measuring the expression of CD69 and CD25. The sulfatide analogs induced activation of iNKT cells in a CD1d-dependent manner. Measurement of cytokines in culture supernatants revealed donor-to-donor variability in responses to sulfatide analogs. However, SAP-3 induced significantly higher IL-2 production compared to control across the six donors tested. To evaluate whether this activation is preserved in cancer patients, we analyzed iNKT cells from glioblastoma patients. SAP-3 effectively activated patient-derived iNKT cells, inducing CD69 and CD25 expression and IL-2 production, consistent with the results observed in healthy donors. Finally, to test the hypothesis that SAP-3 enhances T cell activation and proliferation, we stimulated human PBMC-derived CD8 T cells with the influenza matrix protein (FMP)-derived peptide with or without SAP-3 or α-GalCer. Combining SAP-3 or α-GalCer with FMP did not alter the frequency of cytokine-producing CD8 T cells compared to FMP alone. However, co-administration of SAP-3 or α-GalCer increased the proportion of poly-functional CD8 T cells, producing three cytokines, IFN-γ, IL-2, and TNF-α.Conclusions These data suggest that the sulfatide analog SAP-3 enhances antigen-specific CD8 T cell responses by increasing the proportion of polyfunctional T cells. Importantly, SAP-3 also activated iNKT cells derived from PBMCs of glioblastoma patients, consistent with findings in healthy donors. These results indicate that SAP-3 may serve as a promising novel adjuvant to enhance anti-tumor immune responses, including potential applications in glioblastoma immunotherapy.