This work provides the first use of helium charge transfer dissociation (He-CTD) tandem mass spectrometry (MS/MS) in negative polarity mode. Three sulfated oligosaccharides of natural origin were chosen as representative structures that are difficult to solve by conventional MS/MS approaches. Negative polarity He-CTD provided a full set of structurally informative fragments, which permitted the unambiguous determination of the complete structures of these molecules, including the characterization of labile sulfated functional groups. Despite close structural features, the three molecules led to distinct fragmentation patterns depending on the position of the sulfate group in the heterocycle. The observed fragments showed a consistent radical-initiated mechanism of dissociation, which shares similarities with fragment types produced in electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), or extreme UV photodissociation (XUV-PD). Short times of data collection and the fact that the technique can be affordably implementable in any standard laboratory and with a classical ion trap mass spectrometer were other remarkable characteristics of negative polarity He-CTD.