ABSTRACTThe newly discovered complete ammonia-oxidizing (comammox)Nitrospirahas been identified in different environments, including coastal environments, where salinity is one of the most important factors for the abundance and activity of nitrifiers. Here, we demonstrate the effect of salinity on comammoxNitrospira, canonical ammonia-oxidizing bacteria (AOB), and ammonia-oxidizing archaea (AOA) in the intertidal sediments of the Yangtze River estuary based on microcosm experiments, DNA stable-isotope probing (DNA-SIP), and potential ammonium-oxidation rate (PAR) tests for different groups of ammonia oxidizers with selective inhibitors. During microcosm incubations, the abundance of comammoxNitrospirawas more sensitive to increased salinity than that of other ammonia oxidizers. The results obtained with DNA-SIP heavy fractions showed that the dominant phylotype in clade A.2 (containing genes involved in the adaptation to haloalkaline environments) had high proportions in comammoxNitrospiracommunity under both freshwater (0.06% salinity) and highly saline water (3% salinity) conditions. In contrast, another phylotype of clade A.2 (which lacks these genes) was dominant only under freshwater conditions. The PARs confirmed that comammoxNitrospirapresented greater contributions to nitrification under freshwater conditions with a PAR of 4.37 ± 0.53 mg N·day–1·kg soil–1(54%) than under saline water conditions with a PAR of 0.60 ± 0.94 mg N·day–1·kg soil–1(18%). Moreover, AOA were specific to saline water conditions, whereas AOB were common under both freshwater and saline water conditions (44% and 52%, respectively). The present study provided evidence that salinity markedly affects the activity of comammoxNitrospira,and that the salt sensitivity of different phylotypes varies.IMPORTANCEComplete ammonia oxidation (comammox) is a newly discovered type of nitrification through which ammonia is oxidized to nitrate in an organism. ComammoxNitrospirawere abundantly found in coastal ecosystems and demonstrated high community diversity. Changes in salinity are considered one of the most important factors to comammoxNitrospirain coastal ecosystems; however, reports on the correlation between them remain inconsistent. Therefore, it is critical to experimentally determine the influence of salinity on comammoxNitrospirain the coastal ecosystem. This study demonstrated a clear effect of salinity on the abundance, activity, and relative contribution of different ammonia oxidizers, especially for comammoxNitrospira. To the best of our knowledge, this is the first study demonstrating comammoxNitrospiraactivity at seawater salinities, implying the existence of a salt-tolerant type comammoxNitrospira, despite its activity being much lower than in freshwater conditions. The indicated correlation between the activity of specific comammoxNitrospiraand salinity is anticipated to provide insights into the distribution of comammoxNitrospiraand their potential contributions in estuaries and coastal ecosystems.