Nitrification has been widely applied in wastewater treatment, however gaining more insight into the nitrifiers’ physiology and stress response is necessary for the optimization of nutrient removal and design of advanced processes. Since nitrification initiates with ammonia oxidation performed by ammonia-oxidizing bacteria (AOB), the purpose of this study was to investigate the effects of short-term ammonia starvation on nitrogen uptake and transformation efficiency, as well as the performance of starved nitrifiers under various initial substrate concentrations and pH values. Ammonium deprivation for 3 days resulted in fast ammonium/ammonia accumulation upon nitrogen availability, with a maximum uptake rate of 3.87 mmol gprotein−1 min−1. Furthermore, a delay in the production of nitrate was observed with increasing starvation periods, resulting in slower recovery and lower nitrification rate compared to non-starved cells. The maximum accumulation capacity observed was 8.51% (w/w) independently of the external nitrogen concentration, at a range of 250–750 mg N L−1, while pH significantly affected ammonia oxidizers’ response, with alkaline values enhancing nitrogen uptake. In total, ammonia accumulation after short-term starvation might serve as an important strategy that helps AOB restore their activity, while concurrently it could be applied in wastewater treatment for effective nitrogen removal and subsequent biomass utilization.