Abstract: The structure and fragmentation pathway of silybin were elucidated by electron spray ionization mass spectrometry (ESI-MS）．Quasi-molecular ion peak m/z 481 M-H^- was detected by ESI-MS， and the ions of m/z 453 and m/z 301 were detected by ESI-MS²．The fragment routes of characteristic were discussed on the basis of ESI mass spectra．The full scan MS² spectra of silybin gave characteristic fragment ions formed through cleavage of the C—O—C bond at A-ring and B-ring. The full scan MS³ spectra of fragment m/z 453, m/z 301 was obtained by ion trap mass spectrometry．The characteristic fragmentation was used to prove the structure of m/z 453, m/z 301．