An anti-cancer active substance from Antrodia Cinnamomea, produced from mycelium of Antrodia Cinnamomea by following steps: (1) culturing mycelium of Antrodia Cinnamomea, by liquid fermentation (2) extracting the thus-obtained mycelium with ethanol and concentrating the extracted solution to obtain an ethanol extract (3) dissolving the ethanol extract in water, and extracting with equal volume of ethyl acetate to obtain ethyl acetate extract (4) purifying the ethyl acetate extract by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase to obtain an eluate fraction g and (5) purifying the eluate fraction g by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase to obtain a specific eluate fraction which could be purified further to obtain the anti-cancer active substance from Antrodia Cinnamomea.
