beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated beta3GnT2-deficient (beta3GnT2(-/-)) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in beta3GnT2(-/-) mice. beta3GnT2(-/-) T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3epsilon/CD28 and proliferated more strongly than T cells from WT mice. beta3GnT2(-/-) B cells also showed hyperproliferation on BCR stimulation. Macrophages from beta3GnT2(-/-) mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on N-glycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.