The present study was done to evaluate the prebiotic effect of Lycium barbarum polysaccharide (LBP), its effect on murine fecal microbiota composition and innate immune response. Results showed that LBP supports the growth of selective probiotic bacteria with a maximum of 8.23 (log10 cfu/mL) and 6.62 (log10 cfu/mL) for Lactobacillus acidophilus and Bifidobacterium longum respectively. In vivo studies revealed that the administrations of LBP to mice resulted in an increase in the abundance of the phyla Proteobacteria and Firmicutes, while reducing the ratio of the phylum Bacteroidetes. At the genus level, the administration of LBP stimulated the emergence of some potential probiotic genera (Akkermansia, Lactobacillus, and Prevotellaceae). The concentrations of TGF-β and IL-6 in serum and sIgA in the colon content were enriched significantly after LBP administrations in mice. The thymus index and spleen index of mice treated with LBP displayed significant difference compared to the control group (P < 0.05). These findings suggest that LBP is a good source as a potential prebiotic and can enhance the intestinal microbiota and boost beneficial bacteria levels, modulate innate immune response.