OBJECTIVE To explore the effect and possible mechanism of Xiaoyao San in chronic stress depression mice. METHODS The antidepressant effects of Xiaoyao San (XYS, low-dose 1.40 g·kg^-1, high-dose 2.79 g·kg^-1), mifepristone (1.73 mg·kg^-1) and fluoxetine (2.9 mg·kg^-1) were assessed in chronic unpredictable mild stress (CUMS)-exposed male mice. After the intervention, the depressive-like behaviors of mice were evaluated by open-field test (OFT), sucrose preference test (SPT), forced swimming test (FST), and tail suspension test (TST). Hematoxylin eosin (HE) staining experiment was used to observe the pathological changes of the hippocampal tissue. Enzyme linked immunosorbent assay (ELISA) was used to detect the level of corticosterone (CORT) in serum of mice. Western blot (WB) experiment was used to detect the protein expression of nuclear and cytoplasmic glucocorticoid receptor (GR), phosphorylation GR, FK506 binding protein 51 (FKBP51), brain-derived neurotrophic factor (BDNF) in hippocampal tissue. RESULTS Compared with the control group, mice in the model group showed it was significantly deteriorated behavioral performance (P<0.01) and pathological changes in the hippocampal tissue, indicating the successful establishment of the CUMS model. After XYS treatment, the behavioral performance (P<0.05) of mice and pathologic manifestation of the hippocampal tissue were significantly improved, and the serum CORT level was significantly reduced (P<0.05). In addition, WB examination demonstrated that XYS treatment significantly increased the expression of GR, BDNF, pGR-S211, and nuclear GR in hippocampal tissue (P<0.05), while significantly decreased the expression of FKBP51 and pGR-S226 (P<0.05). CONCLUSION XYS may exert its anti-depressant effects by regulating the expression of FKBP51, phosphorylation GR and BDNF, improving the function of GR and regulating the homeostasis of the hypothalamic-pituitary-adrenal (HPA) axis.