In this article, a steady-state mathematical model was developed and experimentally evaluated to investigate the effect of influent flow distribution and volume ratios of anoxic and aerobic zones in each stage on the total nitrogen concentration of the effluent in the step-feed biological nitrogen removal process. Unlike the previous modeling methods, this model can be used to calculate the removal rates of ammonia and nitrate in each stage and thereby predict the concentrations of ammonia, nitrate, and total nitrogen in the effluent. To verify the simulation results, pilot-scale experimental studies were carried out in a four-stage step feed process. Good correlations were achieved between the measured data and the simulation results, which proved the validity of the developed model. The sensitivity of the model predictions was analyzed. After verification of the validity, the step feed process was optimally operated for five months using the model and the criteria developed for the design and operation. During the pilot-scale experimental period, the effluent total nitrogen concentrations were all below 5mg-L-1, with more than 90% removal efficiency.