Glycosphingolipids are essential components of eukaryotic cell membranes and are involved in the regulation of cell growth, differentiation, and neoplastic transformation. In this work, we have modulated glycosphingolipid levels in CHO cells stably expressing the human serotonin1A receptor by inhibiting the activity of glucosylceramide synthase using (±)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a commonly used inhibitor of the enzyme. Serotonin1A receptors belong to the family of G-protein-coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral, and developmental functions. We explored the function of the serotonin1A receptor under glycosphingolipid-depleted condition by monitoring ligand-binding activity and G-protein coupling of the receptor. Our results show that ligand binding of the receptor is impaired under these conditions although the efficiency of G-protein coupling remains unaltered. The expression of the receptor at the cell membrane appears to be reduced. Interestingly, our results show that the effect of glycosphingolipids on ligand binding caused by metabolic depletion of these lipids is reversible. These novel results demonstrate that glycosphingolipids are necessary for the function of the serotonin1A receptor. We discuss possible mechanisms of specific interaction of glycosphingolipids with the serotonin1A receptor that could involve the proposed ‘sphingolipid-binding domain’.