A combination of tandem mass spectrometry (MS/MS) and hydrogen attachment dissociation (HAD) is a useful method for peptide sequence analysis. In this study, gas-phase fragmentation induced by the attachment of hydrogen to peptides containing disulfide bonds was investigated. Hydrogen attachment induced the cleavage of either the disulfide or N-Cα bond, which competitively occurred during HAD. The disulfide bond cleavage proceeded through an intermediate, which contains a thiyl radical (-S˙) and a thiol group (-SH). In contrast, N-Cα bond cleavage produced an intermediate containing an enol-imine group and α-carbon radical. The intermediate α-carbon radical then attacked the disulfide bond, resulting in a cyclic [z]+ fragment. The counterpart, [c + H]+˙ with a thiyl radical underwent further hydrogen attachment, producing [c + 2H]+. Because both disulfide and N-Cα bonds were cleaved by a single hydrogen attachment event, HAD-MS/MS can provide sequence information for the backbone region in the disulfide loop.