Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragments─similarly to that in ES+─along with y-NH3/z, c, c-NH3/b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages N-terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage N-terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.