A novel strategy to achieve substantial polyhydroxyalkanoates (PHA) accumulation in waste activated sludge (WAS) was developed, which was conducted in a two-sludge system consisted of an anaerobic/anoxic/oxic reactor (AAO-SBR) and a nitrifying reactor (N-SBR), where the nitrifying-sludge was treated by free nitrous acid (FNA). Initially, 0.98 ± 0.09 and 1.46 ± 0.10 mmol-c/g VSS of PHA were respectively determined in the control-SBR and AAO-SBR. When 1/16 of nitrifying sludge was daily treated with 1.49 mg N/L FNA for 24 h, ∼46.5% of nitrite was accumulated in the N-SBR, ∼2.43 ± 0.12 mmol-c/g VSS of PHA was accumulated in WAS in AAO-SBR without deteriorating nutrient removal. However, nutrient removal of control-SBR was completely collapsed after implementing the same FNA treatment. Further investigations revealed that the activity and abundance of nitrite oxidizing bacteria (NOB) was decreased significantly after FNA treatment. Finally, sludge with high PHA level to generate more methane was confirmed.