Manipulation of product heterogeneity was attempted by using yeast extract as nitrogen source in Alternaria alternata S-f6 transformation process of 4'-demethylepipodophyllotoxin. When the nitrogen source of NaNO(3) was replaced by yeast extract, the heterogeneity of biotransformation products was significantly varied from a single product (i.e., 4'-demethylpodophyllotoxone) to four podophyllum derivates. According to the kinetics of 4'-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6, the starting substrate of 4'-demethylepipodophyllotoxin was preferentially transformed to produce 4'-demethylpodophyllotoxone (1) with an oxidation reaction. By the further comparison of products configuration, 4β-caprinoyl-4'-demethylepipodophyllotoxin (3) was produced from 4'-demethylpodophyllotoxone (1) instead of 4'-demethylisopicropodophyllone (2), which might be produced from 4'-demethylpodophyllotoxone (1) with the isomerization of lactone. Finally, 4'-demethylisopicropodophyllone (2) was hydrolyzed to produce 3α-hydroxymethyl-(6, 7)-dioxol-4-one-naphthalene (4). This work shows new information on the 4'-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6 and provides a foundation for further studies on the structural diversification of a bioactive natural lead compound.