© 2020 The Authors Background: Accumulating evidence points to an association between gut microbial abnormalities and depression disorder. The microbiota-gut-brain axis is an emerging target for treating depression using nutritional strategies, considering the numerous limitations of current pharmacological approaches. Here we studied the effect and probable mechanisms of psychobiotic treatment on depression. Methods: Chronically stressed C57BL/6J male mice were administered viable Bifidobacterium breve CCFM1025 for 5 weeks prior to behavioral testing. Brain neurological alterations, serum corticosterone, cytokines levels, fecal microbial composition, and short-chain fatty acid (SCFA) content were measured. In addition, the effect of SCFA on 5-hydroxytryptophan (5-HTP) biosynthesis was investigated in an in vitro model of enterochromaffin cells (RIN14B). Results: CCFM1025 treatment significantly reduced depression- and anxiety-like behaviors. The hyperactive hypothalamic-pituitary-adrenal response, as well as inflammation, were also alleviated, possibly via regulating the expression of glucocorticoid receptors (Nr3c1). Moreover, CCFM1025 also down-regulated the pCREB-c-Fos pathway but increased the expression of brain-derived neurotrophic factor (BDNF). Meanwhile, chronic stress-induced gut microbial abnormalities were restored, accompanied by increased SCFA and 5-HTP levels. The intestinal 5-HTP biosynthesis positively correlated with fecal SCFA and Bifidobacterium breve levels. Conclusions: In summary, Bifidobacterium breve CCFM1025 showed considerable antidepressant-like and microbiota-regulating effects, which opens avenues for novel therapeutic strategies towards treating depression.