Disturbances in gut microbiota contribute to an imbalanced gut-brain axis, which is critical for post-stroke depression (PSD), while the underlying mechanisms remain unclear. The objective of this study was to examine the effects of modifying gut microbiota through antibiotic treatment (ABX) and fecal microbiota transplantation (FMT) on the progression of PSD. The PSD model was established by occluding the middle cerebral artery (MCAO), followed by a four-week isolated housing and restraint stress initiated three days after MCAO. For ABX, the PSD mice received antibiotic water for four weeks. While another group of PSD mice underwent FMT or fluoxetine (FLX) for four weeks. At day 35 post-MCAO, behavioral tests were conducted. Results indicated ABX and FMT significantly altered the composition of intestinal flora caused by PSD, all the treatments markedly attenuated anxiety- and depressive-like behaviors and inflammation in the gut and brain. ABX obviously alleviated PSD-induced disorder of intestinal barrier, decreased mRNA levels of TNF-α, IL-1β and IL-6, and decreased CD4 cells in the colon. While FMT significantly decreased CD8 cells and increased the goblet cells in colon. Furthermore, both ABX and FMT could reduce activated microglia and pro-inflammatory cytokines in the brain, alleviate decreased Nissl's bodies in the hippocampus, and reverse the decreases in 5-HT, Glu and DA in the striatum caused by PSD. Unlike ABX, FMT was similar to FLX. These findings suggest homeostasis of gut microbiota is indispensable for the development of PSD; adjusting the gut microbiota significantly improves PSD with enhanced functions of gut-brain axis.