Abstract: In recent years, the environmental safety problem of neonicotinoid insecticides has become increasingly prominent. As a representative of the new generation of neonicotinoid insecticides, it is of great significance to fully assess the environmental safety of dinotefuran. In this study, the stereoselective adsorption of dinotefuran in water sediment and its effects on the sediment microecosystem were studied by constructing a water-sediment microcosmic system. The results showed that S-dinotefuran had a stronger adsorption capacity in sediment than R-dinotefuran. During the whole exposure period, both R-dinotefuran and S-dinotefuran caused the decrease of urease activity in the sediment. However, the dehydrogenase and catalase activities in the sediment were significantly enhanced under the exposure treatment of either R-dinotefuran or S-dinotefuran. After 28 d of exposure, both R-dinotefuran and S-dinotefuran resulted in significant increases in the abundance of Bdellovibrionota, Hydrogenophaga and Acinetobacter. Additionally, S-dinotefuran significantly inhibited the abundance of Acidobacteriota, Desulfobacterota and Myxococcota compared with R-dinotefuran. S-dinotefuran had a stronger negative effect on the sediment microbial community, resulting in a decrease in microbial community diversity, and promoted the microbial species interaction. The results indicated that the Clostridium_sensu_stricto genus under the Firmicutes phylum played an important role in the degradation of S-dinotefuran, while the microorganisms under the Actinobacteriota phylum played a major role in the degradation of R-dinotefuran. These results are conducive to the environmental safety evaluation of dinotefuran in aquatic ecosystems, and provide theoretical basis for the rational use and pollution remediation of dinotefuran.