The ocean is an important global source of nitrous oxide (N2O), a greenhouse gas that contributes to stratospheric ozone destruction. Bacterial nitrification and denitrification are thought to be the primary sources of marine N2O, but the isotopic signatures of N2O produced by these processes are not consistent with the marine contribution to the global N2O budget. Based on enrichment cultures, we report that archaeal ammonia oxidation also produces N2O. Natural-abundance stable isotope measurements indicate that the produced N2O had bulk delta N-15 and delta O-18 values higher than observed for ammonia-oxidizing bacteria but similar to the delta N-15 and delta O-18 values attributed to the oceanic N2O source to the atmosphere. Our results suggest that ammonia-oxidizing archaea may be largely responsible for the oceanic N2O source.