Sigma-1 receptor (S1R) is a unique multi-tasking chaperone protein in the endoplasmic reticulum. Since S1R agonists exhibit potent antidepressant-like activity, S1R has become a novel target for antidepression therapy. With a rapid and sustained antidepressant effect, ketamine may also interact with S1R. In this study, we investigated whether the antidepressant action of ketamine was related to S1R activation. Depression state was evaluated in the tail suspension test (TST) and a chronic corticosterone (CORT) procedure was used to induce despair-like behavior in mice. The neuronal activities and structural changes of pyramidal neurons in medial prefrontal cortex (mPFC) were assessed using fiber-optic recording and immunofluorescence staining, respectively. We showed that pharmacological manipulation of S1R modulated ketamine-induced behavioral effect. Furthermore, pretreatment with an S1R antagonist BD1047 (3 mg·kg-1·d-1, i.p., for 3 consecutive days) significantly weakened the structural and functional restoration of pyramidal neuron in mPFC caused by ketamine (10 mg·kg-1, i.p., once). Ketamine indirectly triggered the activation of S1R and subsequently increased the level of BDNF. Pretreatment with an S1R agonist SA4503 (1 mg·kg-1·d-1, i.p., for 3 consecutive days) enhanced the sustained antidepressant effect of ketamine, which was eliminated by knockdown of BDNF in mPFC. These results reveal a critical role of S1R in the sustained antidepressant effect of ketamine, and suggest that a combination of ketamine and S1R agonists may be more beneficial for depression patients.© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.