For decades, the conversion of organic nitrogen to dinitrogen gas by heterotrophic bacteria, termed heterotrophic denitrifi-cation, was assumed to be the main pathway of nitrogen loss in natural ecosystems. Recently, however, autotrophic bacteria have been shown to oxidize ammonium in the absence of oxygen , yielding dinitrogen gas. This process, termed anammox, accounts for over 50% of nitrogen loss in marine ecosystems 1–5. However, the significance of anammox in freshwater ecosystems has remained uncertain 6,7. Here, we report the occurrence of anammox hotspots at land–freshwater interfaces of lake riparian zones in North China, using molecular and isotopic tracing technologies. Laboratory incubations measuring anam-mox activity at substrate concentrations no more than 10% of those observed in situ yielded some of the highest potential activities reported for natural environments to date. Potential rates of anammox peaked in sediments sampled from the interface between land and water, as did the abundance of annamox bacteria. Scaling our findings up to the entire lake system, we estimate that interfacial anammox hotspots account for the loss of 103 g Nm −2