Intercellular adhesion molecule-5 (ICAM-5) is a dendrite-specific adhesion molecule, which functions in both the immune and nervous systems. ICAM-5 is the only negative regulator that has been identified for maturation of dendritic spines so far. Shedding of the ICAM-5 ectodomain promotes spine maturation and enhances synaptic activity. However, the mechanism by which ICAM-5 regulates spine development remains poorly understood. In this study, we found that ablation of ICAM5 expression resulted in a significant increase in the formation of synaptic contacts and the frequency of miniature excitatory post-synaptic currents, an indicator of pre-synaptic release probability. Antibodies against ICAM-5 and β1 integrins altered spine maturation. Furthermore, we found that β1 integrins serve as binding partners for ICAM-5. β1 integrins were immunoprecipitated with ICAM-5 from mouse brain and the binding region in ICAM-5 was localized to the two first Ig domains. β1 integrins were juxtaposed to filopodia tips at the early stage of synaptic formation, but as synapses matured, β1 integrins covered the mushroom spines. Loss of β1 integrins from the pre-synaptic sites affected the morphology of the post-synaptic structures. ICAM-5 ectodomain cleavage decreased or increased when the interaction between ICAM-5 and β1 integrins was potentiated or weakened, respectively, using antibodies. These results suggest that the interaction between ICAM-5 and β1 integrins is important in formation of functional synapses.