Sake lees are solid parts filtered from the mash of sake, the traditional rice wine of Japan, which is brewed with Aspergillus oryzae and Saccharomyces cerevisiae. The moisture-holding activity of sake lees has long been recognized in Japan. However, the constituent responsible for this activity has not been elucidated. In this study, we first determined the structure of the glucosylceramides contained in sake lees. The glucosylceramides contained in sake lees were N-2'-hydroxyoctadecanoyl-l-O-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine (d19:2/C18:0h), N-2'-hydroxyoctadecanoyl-l-O-β-D-glucopyranosyl-4,8-sphingadienine (d18:2/C18:0h), N-2'-hydroxyicosanoyl-l-O-β-D-glucopyranosyl-4,8-sphingadienine (d18:2/C20:0h) and N-2'-hydroxyicosanoyl-l-O-β-D-glucopyranosyl-4,8-sphingadienine (d18:2/C22:0h), which corresponded to those of A. oryzae and rice. The glucosylceramide produced by A. oryzae constituted the most abundant species (43% of the total glucosylceramide) in the sake lees. These results will be of value in the utilization of sake lees for cosmetics and functional foods.