Achieving nitrite accumulation via partial denitrification (PD) is a novel technique to supply sufficient nitrite for the anammox process which is used to treat carbon-limited wastewater. Rapid initiation of PD through pH control was developed in this pilot-scale study and the optimum C/N ratio was investigated. The start-up strategy is proposed based on a lab-scale experiment reported in this work in which a high rate of nitrate reduction and an efficient nitrate-to-nitrite transformation ratio (NTR) could be simultaneously achieved at the pH of 9.0. Two pilot-scale reactors (100 L), with one serving as an experimental reactor and the other as a control, were used to determine the pH effect on the start-up of the PD process. In the experimental reactor, the initial pH of 9.0 was applied at the beginning of mixing in each cycle. Results showed that the PD process was rapidly established within 100 cycles with an average NTR of 83.86%, whereas the control reactor achieved PD after 250 cycles. Moreover, the effect of C/N ratios on the PD process was investigated and the result showed that the optimum C/N ratio was 2.5 with both the NTR and nitrate reduction ratio (NRR) exceeding 80%. High-throughput sequencing results demonstrated that Thauera, possibly a functional bacterium for the partial denitrification process, was dominant on the genus level and accounted for over 65% of the microbial community in both PD reactors. The strategy for establishing the partial denitrification process can be easily achieved through long-term stable nitrite accumulation. PD can be integrated with anammox to improve nitrogen removal in an energy-saving mode.