Glycosylation has an important role in regulating properties of proteins and is associated with many diseases. To examine the alteration of serum N-glycans in type 2 diabetes, we used the db/db mouse model. Serum N-glycans were fluorescence-labeled and applied to HPLC. There were reproducible differences in N-glycan profiles between the db/db mouse model and the db/+ control. The structures of the oligosaccharides, which had changed in their amounts, were analyzed by a two-dimensional mapping method, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and exoglycosidase digestion. Those analyses revealed an increase in the N-glycans possessing 1,6-fucose in the serum of db/db mice. The level of 1,6-fucosyltransferase mRNA was increased in the liver of the db/db mice. The ratio of a bi-antennary N-glycan with 1,6-fucose to that without 1,6-fucose in the liver tissue of the db/db mouse was increased relative to the db/+ control. Next, we analyzed the serum N-glycan profile in human subjects with type 2 diabetes and found an increased amount of a bi-antennary N-glycan that had an 1,6-fucose with a bisecting N-acetylglucosamine. In conclusion, the increase in 1,6-fucosylation is a striking change in the serum N-glycans of the db/db mice, whereas the change in the fucosylation in humans with type 2 diabetes was small, albeit statistically significant. It is likely that the change is caused, at least partially, by the increase in the 1,6-fucosyltransferase mRNA level in the liver. The increased 1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 diabetes.