Chronic stress contributes to the pathogenesis of major depressive disorder (MDD). The efficacy of ketamine on the treatment-resistant MDD implies the involvement of glutamatergic dysregulation in its pathophysiology. We recently demonstrated the abnormalities of ubiquitin-proteasome system (UPS) in the pathophysiology of MDD. In the present study, we investigated the involvement of glutamatergic dysregulation by UPS in chronic social defeat stress (CSDS)-induced depression-like behavior. To induce CSDS, the adult C57BL/6J mouse was exposed to aggressor ICR mouse for 10 consecutive days. CSDS reduced the duration of time spent at the interaction zone in the social interaction test. Increase in high potassium-induced release of glutamate was diminished in the prefrontal cortex (PFC) of the stressed mice. Interestingly, ubiquitinated but not total glutamate transporter 1 (GLT-1) was decreased in the PFC of the stressed mice. Protein levels of neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4L) and ubiquitin-conjugating enzyme E2D2 (Ube2d2) were also reduced in the stressed mice. Injection of adeno-associated virus (AAV) expressing shRNA against Nedd4L into the PFC exacerbated the social impairments induced by CSDS. These results suggest that CSDS induces depressive-like behavior and glutamatergic dysfunction, through the decrease of GLT-1 ubiquitination by down-regulation of Ube2d2-Nedd4L pathway. Taken together, GLT-1 ubiquitination could play crucial roles in the pathophysiology of MDD.