The aim of this study was to determine the effect of beta-glycosphingolipids on intra-hepatic natural killer T (NKT) lymphocyte regulatory function and on lymphocyte trapping via alteration of cell membrane lipid rafts. Immune-mediated colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Mice were treated with beta-lactosylceramide (LC), beta-glucosylceramide (GC), beta-galactosylceramide, ceramide, or a combination of both GC and LC (IGL), or solvent alone. Lipid rafts were investigated by fluorescence-activated cell sorting analysis of ganglioside-GM1 and fluorescence microscopy of structure. Administration of beta-glycosphingolipids resulted in an increased intrahepatic/peripheral NKT ratio, increased intrahepatic CD8+ lymphocyte trapping, decreased serum interferon-gamma (IFN-gamma) levels and decreased serum IFN-gamma/interleukin-10 ratio. Administration of GC, LC, or IGL significantly altered the levels of GM1, a key marker of lipid rafts, on NKT regulatory lymphocytes. The immune modulatory effect of beta-glycosphingolipids was associated with increased survival and significant alleviation of colitis as determined by improvement in both the macroscopic and microscopic scores. In conclusion, administration of beta-glycosphingolipids increased NKT regulatory lymphocyte redistribution and intrahepatic CD8(+) T lymphocyte trapping, resulting in alleviation of immune-mediated colitis. The effects of these naturally occurring compounds were associated with modification of the T lymphocyte lipid raft structure, which is a site for immune modulation.