AbstractMatriglycan [-GlcA-β1,3-Xyl-α1,3-]nserves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetylglucosaminyltransferase-1 (LARGE) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show thatProtein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNac-β1,3-GlcNac-β1,4-Man) preceding matriglycan synthesis, is required for LARGE-mediated generation of full-length matriglycan on α-DG (∼150 kDa). In the absence ofPOMK, LARGE synthesizes a very short matriglycan resulting in a ∼90 kDa α-DG in mouse skeletal muscle which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 byPOMKenables LARGE to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.