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Microbial nitrate reduction can be driven by organic carbon oxidation, as well as by inorganic electron donors, such as reduced forms of sulfur and iron. An apparent inverse oxygen isotope fractionation effect was observed during nitrate reduction in sediment incubations from five sampling sites of a freshwater lake, Hongze Lake, China. Incubations with organic and inorganic electron donor additions were performed. Especially, the inverse oxygen isotope effect was intensified after glucose addition, whereas the incubations with sulfide and Fe2+ showed normal fractionation factors. Nitrate reductase encoding genes, napA and narG, were analysed with metagenomics. Higher napA/narG ratios were associated with higher oxygen fractionation factors. The most abundant clade (59%) of NapA in the incubation with glucose was affiliated with Rhodocyclales. In contrast, it only accounted for 8%–9% of NapA in the incubations with sulfide and Fe2+. Differences in nitrate reductases might explain different oxygen isotope effects. Our findings also suggested that large variance of O‐nitrate isotope fractionations might have to be considered in the interpretation of natural isotope records. [ABSTRACT FROM AUTHOR]