The neural basis of anxiety is unclear, which hinders the treatment of anxiety disorders. Here, we found that αCaMKII(+) neurons in the medial prefrontal cortex (mPFC(αCaMKII+)) responded to stressors with increased activity both under physiological conditions and after repeated restraint stress (RRS) in mice. Chemogenetic activation of mPFC(αCaMKII+) neurons ameliorated stress-induced anxiety. A delayed increase in the expression of growth hormone secretagogue receptor 1a (GHS-R1a), the receptor of the peripheral metabolic hormone ghrelin, in mPFC(αCaMKII+) neurons coincided with reduced excitatory synaptic transmission and the development of RRS-induced enhancement of anxiety-related behavior. Virus-mediated GHS-R1a upregulation in mPFC(αCaMKII+) neurons exaggerated the excitation/inhibition (E/I) imbalance and promoted anxiety-related behavior, whereas GHS-R1a knockdown had the opposite effect. We conclude that GHS-R1a signaling contributes to the development of stress-induced anxiety by shaping synaptic activity of mPFC(αCaMKII+) neurons. GHS-R1a may be a new therapeutic target for treating anxiety disorders.