Stimulation of iNKT cells is highly dependent on the structures of the glycolipids presented by CD1d. Furthermore, antigen processing and CD1d loading in lysosomes play central roles in controlling the stimulatory properties of glycolipid antigens. Previously, we determined that substitution at C6'' on alpha-galactosylceramides did not significantly impact stimulatory properties; however, it was not known if substitution at this position influenced lysosomal processing of oligoglycosylceramides. We have prepared a series of mono- and di-galactosylceramides to observe the impact of C6'' substitution on glycosidase truncation of these glycolipids. We found that substitution did not significantly impact glycosidase activity or loading into CD1d.