Enzymatic addition of GalNAc to isotopically labeled IFNα2a produced in Escherichia coli yielded the O-linked glycoprotein GalNAcα-[(13)C,(15)N]IFNα2a. The three-dimensional structure of GalNAcα-IFNα2a has been determined in solution by NMR spectroscopy at high resolution. Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale. Subsequent addition of a Gal unit produced Gal(β1,3)GalNAcα-[(13)C,(15)N]IFNα2a. This extension resulted in a further decrease in the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins.