The high-rate partial denitrification (PD) via effluent residual nitrate controlling by carbon dosage optimization was investigated based on the analysis of microbial mechanism of nitrite accumulation in this study. When the COD/N was changed from 4.0 to 1.8 and the effluent nitrate was above 8.48 mg/L, the nitrate accumulation ratio (NAR) and nitrate removal ratio (NRR) achieved 60 and 90%, respectively. With the electron donor starvation (EDS) strategy, nitrite accumulation was increased, which is related to the reduced utilization of carbon sources. In addition, the rapid increase of Thauera (0.21% to 53.29%) and inhibition of Others and Unclassified (96.93% to 16.99%), and the significantly different expression between reductase genes contributed to nitrite production (narG, 1,727.44 copies/mg) and nitrite reduction (nirS, 208.27 copies/mg; nirK, 203.94 copies/mg) commonly involved in PD start-up and stable operation. Another reactor can be quickly started by controlling effluent residual nitrate within 19 days.