•Intermittent ultrasonic treatment is an effective way to establish nitrite pathway.•Population of nitrite-oxidizing bacteria is successfully suppressed.•Sludge treatment ratio (RS) is identified to be an important operational parameter. Achieving mainstream nitrogen removal via the nitrite pathway (NH4+ → NO2− → N2) is highly beneficial for energy neutral/positive wastewater treatment. Our previous batch assays revealed that ultrasonic treatment can suppress nitrite-oxidizing bacteria (NOB) while enhancing the activity of ammonia-oxidizing bacteria (AOB). Based on this concept, this study investigated the feasibility of applying ultrasonication to achieve the nitrite pathway in mainstream wastewater treatment. Two lab-scale sequencing batch reactors were set-up in parallel and fed with real municipal wastewater. With 100% of the sludge treated every 12 h at a treatment energy input of 0.066 kJ per mg mixed liquor suspended solids, the nitrite pathway was rapidly (within two weeks) established in the experimental reactor with stable effluent nitrite accumulation ratio (NO2−/(NO2− + NO3−)) of above 80% and significantly decreased NOB population. In comparison, the control reactor always possessed the conventional nitrification and denitrification pathway. Economic analysis indicated that energy consumption is too high for practical applications. However, this technology may be used in conjunction with other technologies, whereby this ultrasonic treatment can be used infrequently (e.g. once every few months) when the nitrite pathway becomes unstable.