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• The denitrification rate of urban river sediment feature temporal, spatial and vertical variation. • Urbanization aggravated river pollution, but the denitrification of river sediment can be enhanced. • Denitrification of urban river sediment can remove nearly 20% of the input nitrogen load. Streams and rivers, especially in urban areas, are substantial sinks of bioavailable nitrogen owing to their hydrological connections with terrestrial systems and the intensity of inputs mediated by human activities. Denitrification can account for nitrogen removal in rivers, but its importance in urban rivers has been scarcely studied. This study chose 8 representative rivers in Shanghai, under different level of urbanization, and measured denitrification rates of sediments across a year. The results showed significant differences in temporal, spatial and vertical distributions (p < 0.05). Seasonally, the denitrification rate in spring and winter were lower than in autumn. Spatially, the higher denitrification rate of rivers in residential and industrial areas than that in suburban rivers, which dominated with cultivated land, was weakly related to dissolved oxygen (DO) (p < 0.05, r = −0.380) and ammonium nitrogen (NH 4 +-N) concentration (p < 0.05, r = 0.303) in the water column. Vertically, the surface layer (0–2 cm) accounted for 78.2% of the total denitrification capacity (0–5 cm). The denitrification rates among different depths positively correlated with the extractable nitrate concentration (SNO 3 −-N), the extractable ammonium nitrogen concentration (SNH 4 +-N), the organic carbon concentration (SOC), water content and pH of sediment to varying degrees. In addition, the mean annual denitrification rate of studied rivers was calculated as 2.62 ± 0.420 mg N m−2 h−1, and denitrification contributed nearly 20% of the nitrogen removal, these results underscore the importance of denitrification in nitrogen reduction of urban rivers. [ABSTRACT FROM AUTHOR]