Isoglobotrihexosylceramide (iGb3) has been identified as an endogenous ligand recognized by NKT cells; however, it is a weak agonist compared to the exogenous alpha-galactosylceramide. Modification of the structure of iGb3 might improve its stimulatory activity. In this study, we assessed the stimulating activity of chemically-modified iGb3 analogues on murine hepatic NKT cells. We analyzed the percentage of IFN-gamma- or IL-4-producing cells in hepatic iNKT cell population and found that two chemically-modified iGb3 analogues, especially 4'''-dh-iGb3, induced significantly greater intracellular IFN-gamma+ NKT cells in liver by flow cytometry. In vivo experiments also showed that 4-HO-iGb3 and 4'''-dh-iGb3 are selectively strong inducer for rapid serum IFN-gamma production compared with unmodified iGb3. Comparing the structure of iGb3 and its two iGb3 analogues, 4-HO-iGb3 has an extra hydroxy group on C4, suggesting that the additional hydroxy group of phytosphingosine might augment the stability of the CD1d/glycoceramide complex forming and thereby possibly promote IFN-gamma producing. By further modifying the polysaccharide of glycolipid as did in 4'''-dh-iGb3, we found that 4'''-dh-iGb3 elicited more Th1-biased responses than iGb3 and 4-HO-iGb3. This modification might more strongly strengthen the affinity of the TCR/glycoceramide complex and ultimately polarize iNKT cells to release more Th1 cytokines. Our data suggests that a combination modification on both polysaccharide and sphingosine chain of iGb3 elicits preferential Th1-biased responses.