The human natural killer-1 (HNK-1) glyco-epitope possesses a unique structural feature, a sulfated glucuronic acid attached to lactosamine on the non-reducing termini of glycans. The expression of HNK-1 is temporally and spatially regulated by glucuronyltransferase (GlcAT-P) in the brain. Our previous report showed that mice lacking GlcAT-P almost completely lost HNK-1 expression in the brain and exhibited reduced long-term potentiation (LTP) at hippocampal CA1 synapses. GlcAT-P-deficient mice also showed impaired hippocampus-dependent spatial learning. Although HNK-1 plays an essential role in synaptic plasticity and memory formation, it remains unclear how HNK-1 regulates these functions. In this study, we showed that loss of the HNK-1 epitope resulted in an increase of filopodium-like immature spines and a decrease of mushroom-like mature spines in both the early postnatal mouse hippocampus and cultured hippocampal neurons. However, HNK-1 had no influence on spine density or filopodium formation. Immunofluorescence staining revealed that loss of HNK-1 altered the distribution of postsynaptic proteins such as alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-type glutamate receptor subunit GluR2 and PSD-95 from spine heads onto dendritic shafts without affecting synapse formation, resulting in an increase of shaft synapses in cultured GlcAT-P-deficient neurons. GluR2, a major HNK-1 carrier glycoprotein in postsynaptic density, has the ability to promote spine morphogenesis. Overexpression of GluR2 promoted spine growth in both wild-type and GlcAT-P-deficient neurons, but the increase in GlcAT-P-deficient neurons was lower than that in wild-type neurons. This is the first evidence that HNK-1 is a key factor for normal dendritic spine maturation and is involved in the distribution of postsynaptic proteins.