The absolute configuration of the branched methyl group in ante-iso type side chain moiety on the long chain base of glucocerebroside, HLC-2-A, which was isolated from the sea cucumber Holothuria leucospilota was determined. Oxidation of the glucocerebroside with ozone afforded C13-fragment including the ante-iso moiety. The optically active C13-fragment was synthesized asymmetrically by using the Wittig reaction from chiral synton for comparison with the natural fragment.