This study investigated the rapid start-up of the anaerobic ammonium oxidation (anammox) strategy by inoculating different biomass ratios of denitrifying granular sludge and anammox bacteria. The results demonstrated that two reactors (R1 and R2) were rapidly and successfully started-up on days 25 and 28, respectively, with nitrogen removal rates (NRRs) of 0.70 kg/(m(3).d) and 0.72 kg/(m(3).d) at biomass ratios of 10: 1 (R1) and 50: 1 (R2). The explanation for rapid start-up was found by examining the effect of five different sludge extracellular polymeric substances (EPS) on the activity of anammox bacteria in the batch experiments. Batch experiments results first demonstrated that the denitrification sludge EPS (DS-EPS) enhanced the anammox bacteria activity the most, and NO2-N, NH4+-N removal rates were 1.88- and 1.53-fold higher than the control with optimal DS-EPS volume of 10 mL. The rapid start-up strategy makes possible the application of anammox to practical engineering. Crown Copyright (C) 2016 Published by Elsevier Ltd. All rights reserved.