Objective To investigate the effect of Lactobacillus reuteri CCTCC M 2016546 on chronic unpredictable mild stress (CUMS)-induced depression in rats and explore its potential mechanism. Methods Sixty male Sprague-Dawley (SD) rats were initially randomized into three groups: blank control (n=8), CUMS model (n=43), and prevention (n=9). The other groups except the blank control group received three CUMS stimuli daily. The prevention group was administrated with the L. reuteri CCTCC M 2016546 suspension (1×10⁹ CFU/d) via oral gavage prior to daily stress induction. After 4 weeks, depressive behaviors were assessed by sucrose preference, forced swimming, and open field tests. Successfully modeled rats (n=43) were re-randomized into five groups: model control (n=8), fluoxetine (2.1 mg/kg, n=8), combined therapy (2.1 mg/kg fluoxetine+1×10⁹ CFU/d CCTCC M 2016546, n=9), high-dose CCTCC M 2016546 (5×10⁹ CFU/d L. reuteri CCTCC M 2016546, n=9), and low-dose CCTCC M 2016546 (1×10⁹ CFU/d CCTCC M 2016546, n=9). Rats were administrated with corresponding agents daily for 4 weeks and then subjected to behavioral tests again. The levels of 5-hydroxytryptamine (5-HT) in the hippocampus and adrenocorticotropic hormone (ACTH) and cortisol (CORT) in the serum were quantified via ELISA. Western blotting was performed to determine the protein levels of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB), protein kinase B (Akt), phosphatidylinisitol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and cAMP-response element binding protein (CREB) in the brain tissue. Results Compared with the model control group, all treatment modalities (fluoxetine, combined therapy, and CCTCC M 2016546) alleviated depressive behaviors: increasing sucrose preference (P<0.01, P<0.001), reducing immobility time in suspension (P<0.01, P<0.05), and enhancing horizontal locomotion distance/central zone exploration (P<0.01, P<0.05). Biochemical analyses revealed that treatments reversed the CUMS-induced alterations in 5-HT, CORT, and ACTH levels (P<0.01, P<0.05). Western blotting demonstrated upregulated protein levels of BDNF, PI3K, CREB, TrkB, Akt, and ERK in the fluoxetine and combined therapy groups (P<0.01, P<0.05). High-dose CCTCC M 2016546 elevated Akt, BDNF, CREB, and PI3K levels (P<0.01, P<0.05), while low-dose CCTCC M 2016546 raised Akt and BDNF levels (P<0.01). Prophylactic CCTCC M 2016546 administration primarily enhanced TrkB expression (P<0.05). Conclusion L. reuteri CCTCC M 2016546 ameliorates CUMS-induced depression in rats, potentially by modulating the hypothalamic-pituitary-adrenal axis hyperactivity and activating the PI3K/Akt/CREB/BDNF signaling pathway.