Glycosphingolipids (GSLs) are specialised lipids enriched in the outer leaflet of the plasma membrane (PM) and defects in GSL metabolism underlie a range of devastating diseases. Our research explores the molecular mechanisms linking defective lipid processing to demyelinating neurodegenerative disease. Using CRISPR technology we have generated cell-based models of disease and applied specialised proteomics techniques to show that imbalances in GSL levels change the abundance of specific proteins at the plasma membrane. These proteins play crucial roles in cell adhesion, a process highly relevant to the demyelination phenotype seen in these diseases. One of the altered PM proteins, Neurofascin, is critical for the maintenance of myelin-axon contacts. We show that the Neurofascin isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferrentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of sphingolipidoses.