The majority of human congenital disorders of glycosylation present with clinical features involving disruptions in organ development especially malfunction of the nervous system and muscles. This suggests that protein glycosylation is vital for proper development and function. However, our understanding of the role of glycosylation, particularly in muscle development, is poorly understood. We hypothesise that glycosylation plays an important role in cell-cell communication during muscle differentiation. Here, we performed a proteomic time-course analysis during muscle differentiation in cultured L6 myoblasts/myotubes. Several glycosyltransferases responsible for terminal galactosylation and sialylation were modulated during myogensis including a down-regulation of B4GALT and ST3GAL, and an increase in ST6GAL1. To investigate whether the altered biosynthetic machinery remodels the N-glycome, the muscle differentiation was monitored in cultured L6 cells using glycomics. We observed a decrease in di-galactose-terminating and alpha2,3-linked sialic acid containing glycans, and also an increase in alpha2,6-linked sialic acids containing glycans in end-stage myotubes, confirming a remodelling the N-glycome during muscle development. Importantly, these glycome changes were also observed during in vivo skeletal muscle development of post-natal mice from day 1-21 with the most notable changes being a decrease in alpha2,3- and increase in alpha2,6-sialylation. To investigate the role of sialic acid linkage switching, we knocked down st6gal1 using siRNA in cultured myoblasts prior to differentiation. Immunofluorescence microscopy was performed during myogenesis revealing a defect in both myoblast cell fusion and differentiation compared to scramble siRNA-treated cells. Finally, we performed a quantitative glycoproteomic analysis of muscle differentiation by analysing TMT-labelled glycopeptides by LC-MS/MS employing HCD/EThcD/CID. These data were able to pinpoint changes in specific glycans and localise modification sites on several adhesion molecules and integrins. Taken together, our study will be a valuable resource to further our understanding of the role of glycosylation during muscle development.