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Background: Zuotai, a famous Tibetan medicinal mixture containing metacinnabar, is traditionally used for the purpose of tranquilizing minds and soothing nerves. However, it still lacks substantial experimental data for it to be approved for use. Aim: This study was designed to assess the effects of Zuotai on depressive-like symptoms in a chronic unpredictable mild stress (CUMS) mouse model, and to explore its potential mechanism, particularly the hypothalamic-pituitary-adrenal (HPA) axis pathway. Materials and methods: First, Kunming mice were exposed to the CUMS procedure and simultaneously administered Zuotai or imipramine (positive control) by gavage continuously for 6 weeks. Then, depressive-like behaviors of mice in each group were tested with the sucrose preference test, forced swimming test, tail suspension test, and open field test. Meanwhile, the three key neuroendocrine hormones (corticotropin releasing hormone, adrenocorticotropic hormone and corticosterone) in HPA axis pathway, and the level of the emotion-related monoamine neurotransmitters (5-hydroxytryptamine and norepinephrine) were measured using enzyme-linked immunosorbent assay. Furthermore, total mercury in the hypothalamus and hippocampus were determined using an automatic, direct mercury analyzer. Results: Zuotai or imipramine significantly increased the body weight and the sucrose preference ratio in sucrose preference test, and dramatically improved motor activity in forced swimming test, tail suspension test, and open field test in CUMS mice. Zuotai or imipramine remarkably decreased levels of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone in the HPA axis, and increased levels of 5-hydroxytryptamine and norepinephrine in the serum in CUMS mice. However, a small amount of mercury was deposited in the hypothalamus and hippocampus in Zuotai-treated mice, which may pose a potential risk to the central nervous system. Conclusion: Zuotai has a strong ability to ameliorate depressive-like behaviors in CUMS-treated mice through inhibition of the HPA axis and upregulation of monoamine neurotransmitters. These findings provide new insight into the pharmacological effect of Zuotai on depression.