Eryngiolide A (1), the first member of C20 diterpenoids with the skeleton deriving from a cyclododecane core fused with two γ-lactone units, was isolated from the solid culture of the edible mushroom Pleurotus eryngii. The structure of 1 was elucidated by extensive analysis of NMR spectra. Compound 1 exhibited moderate cytotoxicity against two human cancer lines in vitro.