Glutamatergic synapses constitute a major excitatory neurotransmission system and are regulated by glutamate/glutamine (Gln) cycling between neurons and astrocytes. Gln synthetase (GS) produced by astrocytes plays an important role in maintaining the cycle. However, the significance of GS during synaptogenesis has not been clarified. GS activity and expression significantly increase from postnatal day (PD) 7 to 21, and GS is expressed prior to glial fibrillary acidic protein (GFAP) and is more abundant than GFAP throughout synaptogenesis. These observations suggest that GS plays an important role in synaptogenesis. We investigated this by inhibiting GS activity in neonatal mice and assessed the consequences in adult animals. Lower expression levels of GS and GFAP were found in the CA3 region of the hippocampus but not in the CA1 region. Moreover, synaptic puncta and glutamatergic neurotransmission were also decreased in CA3. Behaviorally, mice with inhibited GS during synaptogenesis showed spatial memory-related impairment as adults. These results suggest that postnatal GS activity is important for glutamatergic synapse development in CA3.