Flammulina velutipes is an edible mushroom and contains a lot of fiber, vitamin B1, B2, niacin and folic acid. This study was conducted to explore the effects of the Flammulina velutipes mushroom on immune cells and immunity. Th1 cytokine productions as IFN-γ, TNF-α, and IL-2 were measured in an activated macrophage by Flammulina velutipes water extract in seven concentrations (0, 5, 10, 50, 100, 250, 500, and 1,000 μg/mL). Also, the splenocyte proliferation index was measured at 48 hours after treatment of the Flammulina velutipes water extract in seven concentrations or mitogen, LPS and ConA. The IFN-γ and TNF-α productions were increased by treatment of the Flammulina velutipes water extract. The TNF-α production was significantly higher in the 50~1,000 μg/mL Flammulina velutipes water extract treated macrophages. The IFN-γ production of macrophages treated with the Flammulina velutipes water extract increased significantly in all groups, and the highest 1000 μg/mL concentration. The splenocyte proliferation index was enhanced when the 10~1,000 μg/mL Flammulina velutipes water extracts were treated compared to the control. These primary results suggest that Flammulina velutipes may enhance the immune function by activation of the macrophage and spleen cell.