<p>Ketamine, a non-competitive N-methyl-D-aspartate acid (NMDA) receptor antagonist, produces rapid and sustained antidepressant actions, but the underlying molecular mechanism remains unclear. The CX₃CL1/CX₃CR1 signaling is closely related to mood disorders, and this study aims to investigate its role in ketamine’s antidepressant actions. We pharmacologically (AZD8797, a selective CX₃CR1 antagonist) and genetically (intra-mPFC microinjection with AAV-CX₃CR1-siRNA) manipulated the CX₃CL1/CX₃CR1 signaling and investigated their effects on ketamine’s antidepressant-like effects in mice treated with corticosterone (Cort), and observed changes in synaptic plasticity in response to these manipulations. We found that 24 h after drug injection, ketamine (10 mg/kg, i.p.) significantly reversed the Cort-induced depression-like behaviors, and inhibited the overexpression of pro-inflammatory cytokines and microglial activation. Ketamine significantly improved the Cort-induced impairment in the dendritic complexity and spine densities. In addition, our ELISA results showed that ketamine significantly inhibited the activation of CX₃CL1/CX₃CR1 signaling, and ketamine attenuated the upregulation of CX₃CR1 and CX₃CL1 expression in Cort-treated HT22 and BV2 cells in vitro. Furthermore, pretreatment with AZD8797 (0.8 mg/kg, i.p., twice a week) completely blocked ketamine’s antidepressant-like behavioral effects and eliminated ketamine-induced enhancement in the synaptic plasticity; intra-mPFC microinjection with AAV-CX₃CR1-siRNA also prevented ketamine’s behavioral effects and beneficial effects on the synaptic plasticity. These findings demonstrated that CX₃CL1/CX₃CR1 signaling-mediated synaptic plasticity played essential roles in ketamine’s antidepressant-like effects, which opened a new door to targeting chemokines to improve depression symptoms.</p><p></p>