The α-subunit of dystroglycan, a member of the dystrophin associated protein complex, binds to extracellular laminin-α2, while its β-subunit interacts with cytoskeletal dystrophin. The exact biological role of dystroglycan, especially during human skeletal muscle development, has not been fully explored. Here, we analysed the distribution and expression characteristics of both dystroglycan subunits and laminin-α2 in primary human skeletal muscle cells. During development, expression levels of all three proteins increased with differentiation. The proteins were relocated from the sarcoplasm to the sarcolemma. The size of α-dystroglycan decreased from 150–220 kDa at the proliferation stage to 100–120 kDa at the late developmental stage. Both α- and β-dystroglycan were involved in forming a complex with their respective partners laminin-α2 and dystrophin/utrophin. Our data show that, during development, cells may employ tightly regulated post-translational species-specific modification to produce different isoforms of α-dystroglycan to participate in appropriate functions.