The hinge region of human immunoglobulin A1 (IgA1), connecting the Fab and Fc regions, is mostly composed of Ser, Thr, and Pro (VPSTPPTPSPSTPPTPSPS); hinge peptide (HP). O-Glycans are naturally attached on only particular five Ser/Thr residues in this region. NMR was employed for analysis of the structural changes in HP upon the glycosylation, especially focusing on the cis/trans isomerization of Pro residues. A series of HP containing C-13,N-15-labeled Pro residues were chemically synthesized and enzymatically glycosylated. The signals from cis and trans forms of the labeled Pro were identified by two-dimensional NMR spectroscopy. Cis/trans ratios of the Pro residues at the C-terminal side of the glycosylated Ser/Thr were reduced from 9-10% to 2-3% by the glycosylation. Thermodynamic analyses indicated that the decrease in the cis/trans ratio was enthalpy-driven. Hydrogen-deuterium exchange experiments and NOE-based structure determination revealed that the intraresidue hydrogen bonds between the amide group of GalNAc and carbonyl oxygen of the peptide backbone of GalNAc-Thr are formed in the major trans conformers, which is consistent with the thermodynamic parameters. These hydrogen bonds largely restrict the psi angle of the peptide backbone and, thereby, should make the trans conformation of the C-terminal Pro residue more stable than the cis conformation. Namely, it is predicted that the restricted y angle causes interresidue steric hindrance for the cis conformation. The appropriate glycosylation of HP probably contributes to the decrease in the unfavorable variety of relative orientations between Fab and Fc in IgA1, through stabilizing the conformation of HP.