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The peptide hormone insulin is central to regulating carbohydrate and fat metabolism in the body by controlling blood sugar levels. Insulin's most active form is the monomer and the extent of insulin oligomerization is related to insulin's activity of controlling blood sugar levels. Electrospray ionization (ESI) of human insulin produced a series of oligomers from the monomer to the undecamer identified using quadrupole ion mobility time-of-flight mass spectrometry. Previous research suggested that only the monomer, dimer and hexamer are native forms of insulin in solution and the range of oligomers observed in the gas phase are ESI artifacts. Here the properties of three distinct oligomer bands, I, II and III, where both the charge state and number of insulin units of the oligomer increase incrementally, were investigated. When Zn(II) was added to the insulin sample the same oligomers were observed, but with 0–6 Zn(II) ions bound to each of the oligomers. The oligomers of bands I, II and III were characterized by comparing their drift times, collision cross-sections, relative intensities, collision-induced dissociation (CID) patterns and relative breakdown energies. Insulin oligomers of band I dissociated primarily by releasing either the 2+ or 3+ monomer accompanied by an oligomer that conserved the mass, charge and Zn(II) of the precursor. Insulin oligomers of bands II and III dissociated primarily by releasing the 2+ monomer accompanied by an oligomer that conserved the mass, charge and Zn(II) of the precursor. Comparison of CID patterns and breakdown energies showed all the oligomers in band II required higher collision energies to dissociate than oligomers in band I, and the oligomers of band III required higher energies to dissociate than the oligomers of band II. These results show that the amount of excess charge on the oligomer in respect to the number of insulin monomers in the oligomer affects their stability.