Objective Genetic variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) encoding an actin-regulatory protein are associated with brain disorders, including intellectual disability and epilepsy. However, specific in vivo neuronal defects and potential treatments forCYFIP2-associated brain disorders remain largely unknown. Here, we characterizedCyfip2heterozygous (Cyfip2(+/-)) mice to understand their neurobehavioral phenotypes and the underlying pathological mechanisms. Furthermore, we examined a potential treatment for such phenotypes of theCyfip2(+/-)mice and specified a neuronal function mediating its efficacy. Methods We performed behavioral analyses ofCyfip2(+/-)mice. We combined molecular, ultrastructural, and in vitro and in vivo electrophysiological analyses ofCyfip2(+/-)prefrontal neurons. We also selectively reduced CYFIP2 in the prefrontal cortex (PFC) of mice with virus injections. Results AdultCyfip2(+/-)mice exhibited lithium-responsive abnormal behaviors. We found increased filamentous actin, enlarged dendritic spines, and enhanced excitatory synaptic transmission and excitability in the adultCyfip2(+/-)PFC that was restricted to layer 5 (L5) neurons. Consistently, adultCyfip2(+/-)mice showed increased seizure susceptibility and auditory steady-state responses from the cortical electroencephalographic recordings. Among the identified prefrontal defects, lithium selectively normalized the hyperexcitability ofCyfip2(+/-)L5 neurons. RNA sequencing revealed reduced expression of potassium channel genes in the adultCyfip2(+/-)PFC. Virus-mediated reduction of CYFIP2 in the PFC was sufficient to induce L5 hyperexcitability and lithium-responsive abnormal behavior. Interpretation These results suggest that L5-specific prefrontal dysfunction, especially hyperexcitability, underlies both the pathophysiology and the lithium-mediated amelioration of neurobehavioral phenotypes in adultCyfip2(+/-)mice, which can be implicated inCYFIP2-associated brain disorders.