Adherens junction–associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1 −/− and Ajap1 W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants.