An anti-cancer active substance from Antrodia Cinnamomea, produced from mycelium of Antrodia Cinnamomea with 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 said ethanol extract in water, and extracting with equal volume of ethyl acetate to obtain ethyl acetate extrct (4) purifying said 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 said 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 said anti-cancer active substance from Antrodia Cinnamomea.
