In this study, Anoectochilus formosanus polysaccharide (AFP) was acquired a via water extraction and alcohol precipitation method. The immunoregulatory activity of AFP was first evaluated on cyclophosphamide (Cy)-treated mice. Galacturonic acid, glucose and galactose were confirmed to be the main components of AFP. AFP demonstrated the ability to stimulate the production of TNF-α and IL-6 in RAW 264.7 macrophages. Not surprisingly, the activation of the NF-κB signaling pathway by AFP was validated via Western blot analysis. Furthermore, AFP could alleviate Cy-induced immunosuppression, and significantly enhance the immunity of mice via increasing the thymus index and body weight, stimulating the production of cytokines (IgA, IgG, SIgA, IL-2, IL-6 and IFN-γ). The improvement in the intestinal morphology of immunosuppressed mice showed that AFP could alleviate Cy-induced immune toxicity. These results have raised the possibility that AFP may act as a natural immunomodulator. Overall, the study of AFP was innovative and of great significance for AFP’s further application and utilization.