The liquid chromatography-electrospray ionization-tandem multi-stage mass spectrometry (LC-ESI-MS(n)) method was developed for the analyses and characterization of steroidal saponins in plant extract from the rhizome of Dioscorea nipponica Makino. The HPLC experiments were performed by means of a reversed-phase C18 column and a binary mobile phase system consisting of water and acetonitrile under gradient elution conditions. Pseudoprotodioscin, methyl protodioscin and dioscin were identified by comparing the retention times, UV spectra and the fragmentation properties of [M-H]- ions with the authentic standards. Four groups of steroidal saponin isomers possessed the [M-H]- ions at m/z 1063, 1045, 901 and 1047, respectively, were observed during the LC-ESI(-)-MS analysis, and three groups of them except the pair of isomers with the [M-H]- ions at m/z 1047 could be differentiated by LC-ESI(-)-MS3. Furthermore, the ESI-MS(n) fragmentation behaviors of the [M+Li]+ ions of pseudoprotodioscin and methyl protodioscin have been investigated, and the observed information helped the structural elucidation of the more abundant isomer with the [M-H]- ion at m/z 1047. As the result, a special sugar sequence of the saccharide chains was observed that not glucose but rhamnose might be connected with the hydroxyl group at C-3 position of the steroidal aglycone.