One-stage autotrophic nitrogen (N) removal, requiring the simultaneous activity of aerobic and anaerobic ammonium oxidizing bacteria (AOB and AnAOB), can be obtained in spatially redox-stratified biofilms. However, previous experience with Membrane-Aerated Biofilm Reactors (MABRs) has revealed a difficulty in reducing the abundance and activity of nitrite oxidizing bacteria (NOB), which drastically lowers process efficiency. Here we show how sequential aeration is an effective strategy to attain autotrophic N removal in MABRs: Two separate MABRs, which displayed limited or no N removal under continuous aeration, could remove more than 5.5 g N/m2/day (at loads up to 8 g N/m2/day) by controlled variation of sequential aeration regimes. Daily averaged ratios of the surficial loads of O2 (oxygen) to NH4+ (ammonium) (LO2/LNH4) were close to 1.73 at this optimum. Real-time quantitative PCR based on 16S rRNA gene confirmed that sequential aeration, even at elevated average O2 loads, stimulated the abundance of AnAOB and AOB and prevented the increase in NOB. Nitrous oxide (N2O) emissions were 100-fold lower compared to other anaerobic ammonium oxidation (Anammox)-nitritation systems. Hence, by applying periodic aeration to MABRs, one-stage autotrophic N removal biofilm reactors can be easily obtained, displaying very competitive removal rates, and negligible N2O emissions.