Streams have an important role in regulating nitrogen (N) transportation from terrestrial ecosystems to downstream waters. Here, we examined how catchment land-use affects potential denitrification rates and the function and composition of denitrifier communities in boreal stream sediments, using stable isotope incubations and qPCR and 454-pyrosequencing targeted on nirS, nirK and nosZ genes. Although land-use influenced the water chemistry as higher nitrite + nitrate (NO x − ) concentration at the agriculture-affected sampling point, sediment organic matter content was found to be the key factor in regulating potential denitrification rates. However, the abundance as well as the diversity and community composition of denitrifying microbes, and genetic N2O production potential (the ratio between nirS + nirK and nosZ gene abundances) were connected to both NO x − and sediment quality. Overall, our results suggest that catchment land-use-driven changes in N and carbon availability affect the denitrification rates, and possibly N2:N2O production ratio, in boreal streams, through altering denitrifier abundance and community composition.