Background Saccharomyces boulardii (Sb) has been reported to have the potential to regulate gut motility. The aim of this experiment was to explore the possible function of Sb in gut hypermotility elicited by repeated water avoidance stress (WAS). Methods Adult male Wistar rats (N = 24) were divided into one of the following three groups: control (C), NS (normal saline) + WAS group (N), and Sb + WAS group (S). A diarrhea-predominant irritable bowel syndrome (IBS-D) model in rats was induced using the WAS method. Gut motility was evaluated by stool pellet expulsion per hour. The contractile activity of the colonic muscle strips was measured using an RM6240 multichannel physiological signal instrument. qRT-PCR and immunohistochemistry were used to assess Toll-like receptor 4 (TLR4) expression in colon tissue. ELISA was used to measure the level of cytokines in the serum and colonic tissue. Also, the microbiota composition was determined using high-throughput 16S rRNA sequencing. Result The results showed that oral Sb decreased the WAS-induced increased defecation and colonic hypermotility in vivo. Furthermore, Sb also decreased the contractile amplitude of colonic circular muscle (CM) and longitudinal muscle (LM) strips in a dose-dependent manner in vitro. Repeated WAS increased TLR4 expression, but Sb reversed it. Sb also reduced interleukin-6 (IL-6), IL-1β, and interferon-γ (IFN-γ) levels in serum and colonic tissue, while increasing IL-10 levels in colonic tissue. Meanwhile, the rats from the NS + WAS group had decreased microbiota diversity and had lower relative abundances of Patescibacteria, Epsilonbacteraeota, Cyanobacteria, and Turicibacter compared with controls. The rats in the Sb + WAS group showed a tendency to increase the relative abundance of Blautia when compared to control rats and had lower relative abundances of Acidobacteria and Anaerostipes compared with the NS + WAS group. Conclusion Our findings demonstrated that Sb improved colonic hypermotility in rats, reversed the high-expression of TLR4 in the colon caused by repeated WAS, modulated cytokines in the colon and serum, and altered the gut microbiota, indicating that Sb may be useful for IBS-D.