ABSTRACT: Salinity strongly influences the physiology and distribution of nitrifying microorganisms, yet the effects of low salinity on these key players in nitrogen cycling remain understudied. This study investigates the impact of hypoosmolarity on different groups of ammonia oxidizers in soil and lake environments, as well as in pure culture isolates. In soil microcosms amended with ammonium, at low salinity levels (~120 µS/cm), which are comparable to values commonly found in pristine terrestrial and aquatic environments, the abundance of ammonia-oxidizing bacteria (AOB), dominated by Nitrosomonas oligotropha, significantly increased. In contrast, the growth of ammonia-oxidizing archaea (AOA), dominated by “Ca. Nitrosotenuis” of the Nitrosopumilaceae family, was stimulated by high salinity (~760 µS/cm). In ammonium-fed lake microcosms, the abundance of AOB, dominated by N. oligotropha, significantly increased under both low (~170 µS/cm) and high salinity (~850 µS/cm) conditions. In the presence of allylthiourea, a bacterial nitrification inhibitor, AOA were found to be sensitive to low salinity in both soil and lake microcosms. Consistently, pure culture studies revealed marked growth inhibition of AOA, especially of members of the Nitrosopumilaceae, under hypoosmolarity, unlike AOB and complete ammonia oxidizers (comammox) strains. Comparative genomic analyses with AOB and comammox, along with transcriptomic studies, suggested that the sensitivity of AOA to hypoosmolarity stress is attributed to a lack of sophisticated osmoregulatory transport systems and their S-layer cell wall structure. Overall, this study highlights the importance of hypoosmolarity as a key factor shaping the ecological niches and distribution of ammonia oxidizers as well as nitrification activities in terrestrial and aquatic environments increasingly affected in their salinities by intensified water cycles due to climate change.