4β-Azido-4-demethyl-4'-O-demethyl-epipodophyllotoxin reacted with phenyliodonium diacetate in MeOH at room temperature to synthesize a novel podophyllotoxin derivative containing a cyclohexadienone group with the original configurations of C2 and C4, which are required for the antitumor activities. The component and structure of the product were confirmed using elemental analysis, high-resolution mass spectrum, 1H and 13C NMR spectra, and infrared spectrum. Furthermore, the fragmentation routes of the product were fully assigned with electron-impact time-of-flight mass spectrometry(EI-TOF MS), which can also be used for the structural elucidation based on the molecular ion at m/z 455 ( 16% ) and three novel characteristic fragment ions at m/z 183 ( 37% ), 425 (7%), and 199 (5%). The cyclohexadienone product is sensitive to acidic media. When it is treated with a trace of acid, another novel derivative containing an orthoquinone moiety is formed, which links with the metabolism of the cancer inhibitors.