Abstract: Iridoid glucosides (IGs) are an important class of widely distributed secondary plant metabolites with a wide range of biological and physiological activities, which can be divided into several sub-classes, such as cyclopentene-type, cyclopentane-type, and epoxytane-type IGs. In this work, the mass spectral fragmentation behaviors of 4 standard epoxytane-type IGs, including picroside I, picroside Ⅱ, sesamoside and catalpol, were studied by negative atmospheric pressure chemical ionization (APCI^-) of quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the MS/MS spectra of M-H^-, not only the frequently occurring neutral losses, such as H₂O, CO₂, CH₃OH and glucosidic units were detected, which confirmed the presence of functional substituents in the structures of the IGs, but also the characteristic ring cleavages of the aglycone moiety were observed, corresponding to ^1,4F^-, ^1,6F^- ions based on accurate mass measurements and the elemental compositions of the product ions, which could give valuable information on the basic structural skeletons. The ^1,4F^- ion resulted from cleavage of the dihydropyran ring with the loss of 3-oxopropanoate, while the emerging of ^1,6F^- ions, which was different from the characteristic fragments ^2,6F^-, ^2,7F^- ions in cyclopentane-type and cyclopentene-type IGs. In addition, ring cleavages of the sugar moieties were also observed, corresponding to ^0,4A₁^-, ^1,4A₁^- and ^2,4A₁^- ions, yielding useful information for their characterization. On the other hand, the mass spectral fragmentation patterns of epoxytane-type IGs in negative ion mode were more characteristic than those in positive ion mode. And the fragmentation pathways of the IGs in APCI^- mode were found to be similar to those in ESI^- mode. Based on the fragmentation patterns of these 4 standard epoxytane-type IGs, 10 IGs were characterized in the extract of Picrohiza scrophulariiflora Pennell using ultra-performance liquid chromatography (UPLC)/Q-TOF MS/MS, of which 8 IGs were unambiguously or tentatively identified as epoxytane-type IGs (picroside Ⅰ, picroside Ⅱ, picroside Ⅲ, picroside Ⅳ, specioside, catalposide, veronicoside and piscroside B) and 2 tentatively identified as cyclopentane type IGs (boschnaloside and mussaenosidic acid). The UPLC/Q-TOF MS/MS method has been demonstrated to be an effective tool in analyzing the IG components in Picrohiza scrophulariiflora Pennell, which provide references for the studies of chemical constituents in the complex matrix and the exploitation of medicinal resources. And in the future, efforts should be made to explain the fragmentation pathways more accurately by combining the calculation of the quantum chemistry at a molecular level.