The insulating layers of myelin membrane wrapped around axons by oligodendrocytes are essential for the rapid conduction of nerve impulses in the central nervous system. To fulfill this function as an electrical insulator, myelin requires a unique lipid and protein composition. Here we show that oligodendrocytes employ a barrier that functions as a physical filter to generate the lipid-rich myelin-membrane sheets. Myelin basic protein (MBP) forms this molecular sieve and restricts the diffusion of proteins with large cytoplasmic domains into myelin. The barrier is generated from MBP molecules that line the entire sheet and is, thus, intimately intertwined with the biogenesis of the polarized cell surface. This system might have evolved in oligodendrocytes in order to generate an anisotropic membrane organization that facilitates the assembly of highly insulating lipid-rich membranes.