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Sulfonamide (SA) antibiotics are ubiquitous pollutants in livestock breeding and aquaculture wastewaters, which increases the propagation of antibiotic resistance genes. Microbes with the ability to degrade SA play important roles in SA dissipation, but their diversity and the degradation mechanism in the field remain unclear. In the present study, we employed DNA-stable isotope probing (SIP) combined with metagenomics to explore the active microorganisms and mechanisms of SA biodegradation in antibiotic-contaminated wetland sediments. DNA-SIP revealed various SA-assimilating bacteria dominated by members of Proteobacteria, such as Bradyrhizobium, Gemmatimonas, and unclassified Burkholderiaceae. Both sulfadiazine and sulfamethoxazole were dissipated mainly through the initial ipso-hydroxylation, and were driven by similar microbes. sadA gene, which encodes an NADH-dependent monooxygenase, was enriched in the 13 C heavy DNA, confirming its catalytic capacity for the initial ipso-hydroxylation of SA in sediments. In addition, some genes encoding dioxygenases were also proposed to participate in SA hydroxylation and aromatic ring cleavage based on metagenomics analysis, which might play an important role in SA metabolism in the sediment ecosystem when Proteobacteria was the dominant active bacteria. Our work elucidates the ecological roles of uncultured microorganisms in their natural habitats and gives a deeper understanding of in-situ SA biodegradation mechanisms., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)