Tricholoma matsutake, a popular food and biopharmaceutical resource in Asia, possesses various pharmacological activities. Although T. matsutake mycelium (TM) may enhance immunity, previous studies, to the best of our knowledge, have been performed on normal animals or cells alone. The present study aimed to evaluate the immunomodulatory activity of TM at doses of 0.3, 1.0 and 2.0 g/kg in cyclophosphamide (CTX)‑induced immunosuppressed mouse models. TM treatment for 2 weeks markedly improved the gain in bodyweight, increased organ indices, reduced hind paw swelling and positively regulated the cytotoxicity of natural killer cells and the proliferation of lymphocytes. These effects are similar to that of thymosin α1 (0.16 mg/kg) which served as the positive control. In CTX‑induced immunosuppressed mice, TM demonstrated marked effects on the modulation of the production of immunoglobulin (Ig)G and IgA, and the levels of interleukin‑2, 6, 10 and 12, interferon‑α and γ and tumor necrosis factor‑α in serum. Compared with CTX mice, the reduced activity of nuclear factor (NF)‑κB in serum and spleen, and phosphorylation of inhibitor of NF‑κB kinase α/β in spleen were observed in TM‑treated mice. Taken together, TM effectively improved immune function in immunosuppressed mice via modulation of ILs and inflammatory factors associated with the NF‑κB signaling pathway.