Abstract: Positive and negative electron spray ionization mass spectrometry (ESI-MS) was used to analyse the chemical structure and MS/MS fragmentation pathway of quercetin. The positive ESI-MS characteristic ions and fragmentation mechanism of quercetin were elucidated and explained by the program of Mass Frontier 3.0. Fragment ions m/z 287, 285, 275, 267, 257 and 229 are existed at the positive ion mode, and the fragment m/z 285 and 275 are further fragmented to form m/z 257 and 229. These ions are the fragmentation characteristics of quercetin in the positive ESI-MS. Fragment ions m/z 283, 273, 257, 245, 229 are the fragmentation characteristics in negative ESI-MS, and the fragment m/z 255 is formed by m/z 283 or m/z 273. The fragmentation pathway in positive and negative ESI-MS is similar, but the negative ESI-MS mode provides less fragment ions. These results provide the very important information for the drug metabolic research and the structural modification of quercetin.