To investigate the regulatory effects of β-nicotinamide mononucleotide (β-NMN) on cyclophosphamide (CTX)-induced immunosuppression in mice, the experimental mice were divided into the following groups: a blank control group, a CTX model group, β-NMN intervention groups (low, medium, and high doses), and a positive drug control group. Parameters were measured, including body weight, spleen and thymus indices, carbon clearance capacity, phagocytic activity, antibody levels, natural killer (NK) cell activity, serum nicotinamide adenine dinucleotide (NAD+) content, as well as inflammatory cytokine levels and CD38/Slc12a8 protein expression in the spleen. The results showed that low, medium and high doses of β-NMN significantly increased the body weight (2.95, 3.32, 4.13 g), spleen index (5.39, 5.01, 5.40 g•kg-1), thymus index (3.12, 3.15, 3.17 g•kg-1), phagocytic rate (27.50%, 38.20%, 41.10%), antibody levels (7.70, 8.70, 10.90 plaques/105 splenocytes), NK cell activity (28.10%, 40.72%, 47.45%), and NAD+ content (7.89, 13.61, 25.12 nmoL•μL-1) in the intervention groups compared to the model group (P<0.05). Furthermore, the high-dose β-NMN group was found to significantly elevate Il10 and Slc12a8 mRNA levels (1.60, 1.97) and significantly downregulate Il1b, Il6, Tnfa, and Cd38 mRNA levels (0.74, 0.66, 0.88, 0.60) (P<0.05). These results indicate that CTX-induced immunosuppression in mice can be effectively alleviated by β-NMN, and the underlying mechanism may involve the CD38/NAD+ metabolic pathway, thereby providing a theoretical foundation for the potential application of β-NMN in immunomodulatory therapeutics and the food industry.