Bacillus coagulanshas a potential role in improving intestinal injury. However, the specific mechanism is still unclear. In this study, the protective effect ofB. coagulansMZY531 on intestinal mucosa injury in cyclophosphamide (CYP)-induced immunosuppressed mice were investigated. The results indicated that the immune organ (thymus and spleen) indices ofB. coagulansMZY531 treatment groups were significantly increased compared to the CYP group.B. coagulansMZY531 administration promotes the expression of immune proteins (IgA, IgE, IgG, and IgM).B. coagulansMZY531 could upregulate the ileum levels of IFN-γ, IL-2, IL-4, and IL-10 in immunosuppressed mice. Moreover,B. coagulansMZY531 restores the villus height and crypt depth of the jejunum and alleviates injury of intestinal endothelial cells caused by CYP. Furthermore, the western blotting results showed thatB. coagulansMZY531 ameliorated CYP-induced intestinal mucosal injury and inflammatory via up-regulates the ZO-1 pathway and down-regulates the expression of the TLR4/MyD88/NF-κB pathway. After treatment withB. coagulansMZY531, the relative abundance ofFirmicutes phylumwas dramatically increased, as well as the genera ofPrevotellaandBifidobacterium, and reducing harmful bacteria. These findings suggested thatB. coagulansMZY531 has a potential immunomodulatory activity on chemotherapy-induced immunosuppression.