It was previously revealed [Yamaguchi, H., Nishiyama, T., and Uchida, M. (1999) J. Biochem. 126, 261-265] that N-glycans of both the high-mannose and complex types have binding affinity for aromatic amino acid residues. This study shows that free N-glycans protect proteins from protease digestion through their binding affinities for the aromatic amino acid residues exposed on protein molecules. Protease digestion of bovine pancreatic RNase A and bovine a-lactalbumin was depressed in solutions (1 mM or so) of free N-glycans of both the high-mannose and complex types. The increasing order of the protective effects of the N-glycans paralleled that of their affinities for aromatic amino acid residues; and the presence of aromatic amino acids practically abolished the protective effects of the N-glycans. The N-glycans also depressed the protease digestion of metallothionein, an aromatic amino acid-free protein, in agreement with the observation that the N-glycans also interact with the solvent-exposed aromatic amino acid residues of the proteases. Thus it seems probable that the N-glycans protect proteins from protease digestion by steric hindrance attributable to their binding affinity for the solvent-exposed aromatic amino acid residues of both substrate proteins and proteases.