Nitrogen (N) retention in streams is an important ecosystem service that may be affected by the widespread burial of streams in stormwater pipes in urban watersheds. We predicted that stream burial suppresses the capacity of streams to retain nitrate (NO₃ ⁻) by eliminating primary production, reducing respiration rates and organic matter availability, and increasing specific discharge. We tested these predictions by measuring whole-stream NO₃ ⁻ removal rates using ¹⁵NO₃ ⁻ isotope tracer releases in paired buried and open reaches in three streams in Cincinnati, Ohio (USA) during four seasons. Nitrate uptake lengths were 29 times greater in buried than open reaches, indicating that buried reaches were less effective at retaining NO₃ ⁻ than open reaches. Burial suppressed NO₃ ⁻ retention through a combination of hydrological and biological processes. The channel shape of two of the buried reaches increased specific discharge which enhanced NO₃ ⁻ transport from the channel, highlighting the relationship between urban infrastructure and ecosystem function. Uptake lengths in the buried reaches were further lengthened by low stream biological NO₃ ⁻ demand, as indicated by NO₃ ⁻ uptake velocities 17-fold lower than that of the open reaches. We also observed differences in the periphyton enzyme activity between reaches, indicating that the effects of burial cascade from the microbial to the ecosystem scale. Our results suggest that stream restoration practices involving “daylighting” buried streams have the potential to increase N retention. Further work is needed to elucidate the impacts of stream burial on ecosystem functions at the larger stream network scale.