Ribitol phosphate modifications to the core M3 O-mannosyl glycan are important for the functional maturation of α-dystroglycan. Three sequentially extended partial structures of the core M3 O-mannosyl glycan including a tandem ribitol phosphate were regio- and stereo-selectively synthesized: Rbo5P-3GalNAcβ, Rbo5P-1Rbo5P-3GalNAcβ, and Xylβ1-4Rbo5P-1Rbo5P-3GalNAcβ (Rbo5P, d-ribitol-5-phosphate; GalNAc, N-acetyl-d-galactosamine; Xyl, d-xylose). Rbo5P-3GalNAcβ with p-nitrophenyl at the aglycon part served as a substrate for ribitol phosphate transferase (FKRP, fukutin-related protein), and its product was glycosylated by the actions of a series of glycosyltransferases, namely, ribitol xylosyltransferase 1 (RXYLT1), β1,4-glucuronyltransferase 1 (B4GAT1), and like-acetyl-glucosaminyltransferase (LARGE). Rbo5P-3GalNAcβ equipped with an alkyne-type aglycon was also active for FKRP. The molecular information obtained on FKRP suggests that Rbo5P-3GalNAcβ derivatives are the minimal units required as the acceptor glycan for Rbo5P transfer and may serve as a precursor for the elongation of the core M3 O-mannosyl glycan.