Coordinated development of excitatory and inhibitory synapses is essential for higher brain function, and impairment in this development is associated with neuropsychiatric disorders. In contrast to the large body of accumulated evidence regarding excitatory synapse development, little is known about synaptic adhesion and organization mechanisms underlying inhibitory synapse development. Through unbiased expression screens and proteomics, we identified immunoglobulin superfamily member 21 (IgSF21) as a neurexin2α-interacting membrane protein that selectively induces inhibitory presynaptic differentiation. IgSF21 localizes postsynaptically and recruits axonal neurexin2α in a trans-interaction manner. Deleting IgSF21 in mice impairs inhibitory presynaptic organization, especially in the hippocampal CA1 stratum radiatum, and also diminishes GABA-mediated synaptic transmission in hippocampal CA1 neurons without affecting their excitatory synapses. Finally, mice lacking IgSF21 show a sensorimotor gating deficit. These findings suggest that IgSF21 selectively regulates inhibitory presynaptic differentiation through interacting with presynaptic neurexin2α and plays a crucial role in synaptic inhibition in the brain.Molecular mechanisms regulating the development of inhibitory synapses are poorly understood. Here the authors show that IgSF21 interacts with neurexin2α to induce presynaptic differentiation of inhibitory synapses, and that mice lacking IgSF21 exhibit deficits in inhibitory synaptic transmission.