IRSp53 is an adaptor protein that acts downstream of Rac and Cdc42 small GTPases and is implicated in the regulation of membrane deformation and actin filament assembly. In neurons, IRSp53 is an abundant postsynaptic protein and regulates actin-rich dendritic spines; however, its in vivo functions have not been explored. We characterized transgenic mice deficient of IRSp53 expression. Unexpectedly, IRSp53(-/-) neurons do not show significant changes in the density and ultrastructural morphologies of dendritic spines. Instead, IRSp53(-/-) neurons exhibit reduced AMPA/NMDA ratio of excitatory synaptic transmission and a selective increase in NMDA but not AMPA receptor-mediated transmission. IRSp53(-/-) hippocampal slices show a markedly enhanced long-term potentiation (LTP) with no changes in long-term depression. LTP-inducing theta burst stimulation enhances NMDA receptor-mediated transmission. Spatial learning and novel object recognition are impaired in IRSp53(-/-) mice. These results suggest that IRSp53 is involved in the regulation of NMDA receptor-mediated excitatory synaptic transmission, LTP, and learning and memory behaviors.