This paper reports the first comparative study of the gas-phase fragmentation chemistry of monensin in negative ion mode electrospray and nanoelectrospray tandem mass spectrometry. The fragmentation was observed to occur at lower energies in nanoelectrospray than electrospray. The major product ions are proposed to be formed via an initial neutral elimination of methanol followed be subsequent fragmentation. The low-mass product ions were observed at the same m/z for both monensin A and B.