Abstract: The purpose of this study was to investigate the enhancing effect of Lactobacillus acidophilus LA15 on cyclophosphamide (CTX)-induced immunosuppression in BALB/c mice. The in vivo immunosuppression model was established by administering CTX through injection, and the immunomodulatory effects were assessed by measuring murine phenotypic indicators, immune markers, and gut microbiota. The results indicated that after induction by CTX, mice in the CTX group exhibited decreased body weight, thymus index, spleen index, immunoglobulin levels, and cytokine content. In contrast, LA15 significantly enhanced the thymus index, spleen index (P<0.05), relieve the swelling and inflammatory injury of small intestinal. The intestinal immunoglobulin A (sIgA) content in the LA15 group was 486.39 μg/mL, which was significantly higher than that in the model control group (294.72 μg/mL) (P<0.01), and the levels of TNF-α, IL-6, IL-10, IL-1β, IL-17 increased. 16S rDNA gene sequencing revealed that LA15 reduced the ratio of Firmicutes/Bacteroidetes, increased the relative abundance of Alloprevotella and Lachnospiraceae, and decreased the relative abundance of Desulfurvibrio. Therefore, LA15 could alleviate CTX-induced immunosuppression, reshape the gut microbiota, and was considered a potential immunostimulant for the treatment of immune suppression disorders.