Abstract: In this study, eight potential functional strains were selected to interfered with RAW264.7 murine macrophages and human peripheral blood mononuclear cells (PBMCs). Then, changes in phagocytic activity of RAW264.7 cells and natural killer (NK) cell activity were detected and the screened potential probiotics were further intervened in BALB/c mice to explore their immunomodulatory efficacy in vivo. In cell experiments, the results showed that the intervention of different strains significantly increased the phagocytic activity of RAW264.7 cells (P<0.05), while Bifidobacterium bifidum FL-228.1 and Lacticaseibacillus rhamnosus FN518 significantly increased NK cell activity (P<0.05). In general, Bifidobacterium bifidum FL-228.1 showed the best in vitro performance for further in vivo studies. The intake of Bifidobacterium bifidum FL-228.1 promoted thymus development and significantly increased the phagocytic activity of peritoneal macrophages, NK cell activity in spleen, serum IgG content, splenic lymphocyte transformation and the expression of antimicrobial peptide-related genes Cryptdin-4 and CRAMP in mice (P<0.05), but had no significant effect on the serum cytokines TNF-α, IL-10, IL-12, IFN-γ and the expression of antimicrobial peptide-related gene RegIII-γ (P>0.05). In conclusion, Bifidobacterium bifidum FL-228.1 can improve innate immune function and have a more comprehensive effect on the immune system by regulating immune cell activity, cytokine expression and mRNA levels of immune molecules related to antimicrobial peptides.