Some cytokines, such as interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), produced by lymphocytes might play an important role in anti-tumor immunity and their production is possibly suppressed by cancer. Amelioration of the suppression of cytokine production might contribute to cancer control. Ganoderma lucidum polysaccharides (Gl-PS), a versatile group of a component of G. lucidum and one with various bioactivities, might have this potential. In this study, analyses including reverse transcription and the polymerase chain reaction (RT-PCR), immunocytochemistry and Western blot were used to test the effects of Gl-PS on the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes by incubating Gl-PS with mouse splenic mononuclear lymphocytes in the presence of B16F10 cell culture supernatant following activation by phytohemagglutinin. The RT-PCR, immunocytochemistry and Western blot assays showed that the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes was suppressed by B16F10 cell culture supernatant at both the mRNA and protein levels, whereas the suppression was fully or partially ameliorated by Gl-PS. The amelioration by Gl-PS against the suppression of the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes by B16F10 cell culture supernatant might contribute to cancer control.