Abstract: The formation of substituted 2-hydroxypyridine cations in the fragmentation of protonated N-pyridinyl benzamides was studied by high-resolution electrospray ionization-high energy collision dissociation (HCD) tandem mass spectrometry in combination with isotopic labeling experiments and density functional theory (DFT) calculation. As shown by the results, under the acition of HCD the amide group of protonated N-pyridinyl benzamides was first tautomerized to iminol group. Then the iminol hydroxyl group was rearranged to the C² atom of the pyridine ring through a intramolecular 1,3-OH^- shift. Finally, a substituted 2-hydroxypyridine cation was obtained by cleavage of C²-N³ bond with neutral loss of substituted benzonitrile.