Artificial microbial nitrogen (N) cycle hotspots in the plant-bed/ditch system were developed and investigated based on intact core and slurry assays measurement using isotopic tracing technology, quantitative PCR and high-throughput sequencing. By increasing hydraulic retention time and periodically fluctuating water level in heterogeneous riparian zones, hotspots of anammox, nitrification, denitrification, ammonium (NH4+) oxidation, nitrite (NO2-) oxidation, nitrate (NO3-) reduction and DNRA were all stimulated at the interface sediments, with the abundance and activity being about 1-3 orders of magnitude higher than those in nonhotspots. Isotopic pairing experiments revealed that in microbial hotspots, nitrite sources were higher than the sinks, and both NH4+ oxidation (55.8%) and NO3- reduction (44.2%) provided nitrite for anammox, which accounted for 43.0% of N-loss and 44.4% of NH4+ removal in riparian zones but did not involve nitrous oxide (N2O) emission risks. High-throughput analysis identified that bacterial quorum sensing mediated this anammox hotspot with B.fulgida dominating the anammox community, but it was B. anammoxidans and Jettenia sp. that contributed more to anammox activity. In the nonhotspot zones, the NO2- source (NO3- reduction dominated) was lower than the sink, limiting the effects on anammox. The in situ N2O flux measurement showed that the microbial hotspot had a 27.1% reduced N2O emission flux compared with the nonhotspot zones.