The nature of the NO-bond in the N-oxide group was investigated by means of combined theoretical calculations (including QTAIM and NBO approaches) and statistical analyses of the contents of crystal structure databases. The N-O bond in the N-oxide group should be classified as the NO donating bond with an important contribution of ON back-donation (of the π-electron type, when available). The visualization of the Laplacian of electron density in the region of an oxygen valence sphere suggests the presence of two lone pairs for the imine-N-oxide group (characterized by effective ON back-donation). A detailed bonding analysis performed by means of natural resonance theory indicates that the N→O bond is of an order of magnitude clearly greater than 1. In addition, the stability of the N→O bond in various N-oxides was estimated. The analyses of the hydrogen- and halogen-bonded complexes of the N-oxides reveal strong Lewis basicity of the N-oxide group. The formation of H- and X-bonding leads to N→O bond elongation due to its structural, topological and spectroscopic characteristics. Moreover, in pyridine-N-oxide, the electron-withdrawing -NO2 group additionally stabilizes the N→O bond, whereas the opposite effect can be observed for the electron-donating-NH2 substituent. This is due to a substituent effect on the π-type ON back-donation. As a result, the oxygen atom in pyridine-N-oxide may change its availability during intermolecular interaction formation, as revealed in the interaction energy, which changes by about half of the estimated total interaction energy.