Abstract Background Ninjin’yoeito (NYT), a traditional Japanese Kampo medicine, improves the depression and anxiety in humans and animals, rendering it a novel therapeutic option for post-stroke depression (PSD). Furthermore, physical exercise is an important nonpharmacological therapy for major depressive disorder. The components of NYT or exercise exert antidepressant effects through the increased expression of neurotrophic factors and reduced neuroinflammation in the brain. However, the mechanisms underlying the antidepressant effects of NYT and exercise in PSD remain unclear. Therefore, we examined the effects of NYT and physical exercise in a rat model of PSD. Methods Rats were divided into five groups: PSD, PSD with NYT, PSD with exercise (Ex), PSD with NYT and exercise (NYT + Ex), and control (sham). PSD was induced by the microinjection of endothelin-1 into the left medial prefrontal cortex and chronic unpredictable mild stress 3 days per week. A diet containing 3% NYT was administered to rats one day after stroke induction. Exercise was conducted using a motorized treadmill for three days per week, starting three days after the stroke. The therapeutic interventions lasted for four weeks. Serum corticosterone levels, depression-like behavior, and hippocampal pathophysiology, including the expression of brain-derived neurotrophic factor (BDNF), precursor BDNF (proBDNF), doublecortin (DCX), NeuN, glial cell activation, and tumor necrosis factor-α (TNF-α), were examined. Results Serum corticosterone levels were lower in the treatment group than those in the PSD group. Notably, serum corticosterone levels were significantly lower in the NYT group than those in the PSD group. BDNF expression in the CA1 region was significantly higher in the Ex group than that in the PSD group. The NYT + Ex group showed a significantly higher hippocampal BDNF/proBDNF ratio than the other groups. DCX and NeuN expression levels were significantly higher in the NYT + Ex group than those in the NYT and PSD groups. Hippocampal glial cell activation and TNF-α expression increased in the PSD group and decreased in the intervention groups. Conclusions NYT ameliorates serum corticosterone levels and hippocampal neuroinflammation in PSD. Additionally, this study suggested that NYT, together with exercise therapy, may improve neurogenesis, the BDNF/proBDNF ratio, and neuroinflammation in the hippocampus in PSD. Clinical trial number Not applicable.