Natural killer T (NKT) cells are a unique subset of thymus-derived T cells that recognize glycolipids presented by the CD1d molecule expressed on antigen-presenting cells. The most studied agonistic antigen of NKT cells is alpha-galactosylceramide (αGC). It has been shown that structural features of lipid antigens distinctly induce NKT cell responses, which emphasizes the importance of understanding the relationship between structural characteristics of lipid antigens and context-dependent NKT cell responses. In a previous study, we showed that human gut symbiont Bacteroides fragilis (B.fragilis) uniquely produces αGCs (BfaGC), which can normalize the overproliferation of neonatal colonic NKT cells in germ-free mice, and that chemically synthesized structural isomers of BfaGCs induce structure-specific NKT cell responses. In this study, to further investigate the essential structural moiety of BfaGC for the unique immunomodulatory activity, we synthesized a panel of BfaGC structural variants by modifying their lipid chain structures and functional groups. We have examined how the variants diRerently impact NKT cell responses by analyzing eRector cytokine production, NKT cell proliferation, relative binding aRinities to CD1d, and preferential antigenic recognition by specific TCR Vβ repertoires. We identified critical features and functional groups on the BfaGC structure leading to immunomodulatory outcomes. It is expected that our findings may advance further development of lipid antigens to fine-tune context-specific NKT cell responses in immunological diseases.