The gas-phase reactivity of Ni+(urea) has been investigated by means of mass spectrometry techniques and density functional calculations. The major fragmentations observed in the MIKE spectrum of [Ni-urea]+ correspond to the loss of CO, NH3, and HNCO. The electrospray MS/MS spectrum shows also these fragmentations; however, an additional intense peak is detected matching to the elimination of water. To explain the differences observed in the reactivity under FAB or ESI conditions, several pathways leading to the experimental fragmentations have been considered theoretically. The exploration of the potential energy surfaces has shown that, although the elimination of NH3 mainly arises from the noninsertion mechanisms, the elimination of CO and HNCO arises exclusively from the insertion mechanisms. Elimination of water shows larger barriers, but under electrospray conditions, the presence of the solvent can reduce these energy barriers leading to isomerizations of the [Ni-urea]+ complex in the source region. The results obtained have been also compared to those previously reported for Cu+. Calculations show that the most stable Ni+-urea complex has the Ni+ cation interacting with the oxygen, with the computed binding energy being 66.3 kcal/mol.