Hydrous defects in diopside (CaMgSi2O6) play an important role in the water budget of the Earth’smantle. Related OH-stretching modes lead to a variety of infrared absorption bands observed in natural or ex-perimental samples. In the present study, we report new low-temperature infrared spectra of reference naturaldiopside samples in the OH-stretching range. In parallel, the structure and vibrational properties of a series ofOH-bearing defects in diopside are theoretically determined at the density functional theory level. The infraredspectra make it possible to resolve additional bands in the region above 3600 cm−1and reveal that their anhar-monic behavior differs from that of the bands at lower frequency. A comparison of theoretical results with ex-perimental data makes it possible to propose atomic-scale geometries corresponding to observed OH-stretchingbands. It confirms that the bands observed at 3620–3651 cm−1are related to M3+ions substituted for Si intetrahedral sites, while the 3420 cm−1band is associated with the Na+for Ca2+substitution. In both cases,H+incorporation compensates the charge deficit due to the heterovalent substitution. The other major mecha-nism of water incorporation in diopside relates to the charge compensation of cationic vacancies, among whichCa vacancies play a central role. The 3357 cm−1band corresponds to doubly protonated Ca vacancies in purediopside. In experimental diopside-bearing trivalent cations, the bands at 3432–3460 cm−1correspond to singlyprotonated Ca vacancies with a nearby octahedral M3+ion, while the 3310 cm−1band likely involves a moreremote charge compensation by M3+ions. More complex defects associating Ca vacancies with tetrahedral M3+and octahedral Ti4+ions are proposed for the bands observed between 3500 and 3600 cm−1in natural diopside.The Fe2+for Mg2+and Fe2+for Ca2+substitutions are also found to affect nearby OH-bearing defects, causinga shift and broadening of OH stretching bands in chemically more complex diopside samples.