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There is still a lack of understanding of H2S formation in agricultural waste, which leads to poor odour prevention and control. Microbial sulfate reduction is a major process contributing to sulfide formation in natural and technogenic environments with high sulfate and low oxygen concentration. Agricultural waste can be considered a low-sulfate system with no obvious input of oxidised sulfur compounds. The purpose of this study was to characterise a microbial community participating in H2S production and estimate the microbial sulfate reduction rate (SRR) in manure slurry from a large-scale swine finishing facility in Western Siberia. In a series of manure slurry microcosms, we identified bacterial consortia by 16S rRNA gene profiling and metagenomic analysis and revealed that sulfate-reducing Desulfovibrio were key players responsible for H2S production. The SRR measured with radioactive sulfate in manure slurry was high and comprised 7.25 nmol S cm−3 day−1. Gypsum may be used as a solid-phase electron acceptor for sulfate reduction. Another plausible source of sulfate is a swine diet, which often contains supplements in the form of sulfates, including lysine sulfate. Low-sulfur diet, manure treatment with iron salts, and avoiding gypsum bedding are possible ways to mitigate H2S emissions from swine manure.