AbstractThe colonization ability of Limosilactobacillus fermentum DALI02 and the promoting effect of fermented prebiotics have been studied. This study aims to evaluate the systemic immunomodulatory effects of DALI02 and DALI02 + Prebiotics on cyclophosphamide‐induced immunosuppressed rats. We found that DALI02 and DALI02 + Prebiotics, especially DALI02 + Prebiotics, exhibited significant restorative effects on the immunocompromised state in rats (p < .05). Specifically, both of them promoted the recovery of body weight and immune organ function, enhanced the proliferative capacity of immune cells, and effectively reduced the levels of interleukin 6 (IL‐6) and tumor necrosis factor‐α (TNF‐α). Furthermore, both of them significantly reduced the levels of lipopolysaccharide (LPS) and D‐lactic acid in the blood (p < .05). Principal coordinate analysis (PCoA), principal component analysis (PCA), and unsupervised cluster analysis revealed that DALI02 and DALI02 + Prebiotics group were more similar to the blank group at the genus level of the gut microbiota. At the level of short‐chain fatty acids (SCFAs), DALI02 + Prebiotics and blank group belonged to Cluster 3. These results suggested that the intervention with DALI02 and DALI02 + Prebiotics effectively modulated the structure of the gut microbiota, and DALI02 + Prebiotics restored the dysregulation of SCFAs. In summary, DALI02 and DALI02 + Prebiotics possess immunomodulatory functions, with the latter showing superior effects.