Our results further define the functional O-mannosylated glycan structure and indicate that B4GAT1 is involved in the initiation of the LARGE-dependent repeating disaccharide that is necessary for extracellular matrix protein binding to O-mannosylated α-dystroglycan that is lacking in secondary dystroglycanopathies. Here, B4GAT1 is linked to neuromuscular disease caused by qualitative or quantitative defects of alpha-dystroglycan.