We evaluated the effects of seven mushroom extracts (Grifola frondosa, Pholiota nameko, Panellus serotinus, Hypsizygus marmoreus, Pleurotus cornucopiae, Armillaria mellea, and Flammulina velutipes) on cytotoxic activity and cytokine production of lamina propria leukocytes (LPLs) isolated from rat small (S) and large (L) intestinal mucosa. Boiling water extracts from seven species of mushrooms showed no direct cytotoxicity against the YAC-1 target cells. However, prominent increases of cytotoxicity were observed in S-and L-LPLs co-cultured with P. serotinus extract. Cytokine production (TNFα, IFNγ, IL-12 p70, and IL-4) of S-and L-LPLs was stimulated in response to P. cornucopiae extract. Mushroom extracts contribute to target cell adhesion and/or cytokine production in the effector cells. The promotion of cytotoxic activity in S-and L-LPLs was not necessarily related to β-glucan content of the mushroom.