Free nitrous acid (FNA) has been identified to be a ubiquitous inhibitor of a wide range of microorganisms, including bacteria involved in wastewater treatment. The FNA-induced inhibition on the anoxic (nitrite as electron acceptor) metabolism of denitrifying poly-phosphate accumulating organisms (DPAOs) was investigated using sludge from a sequencing batch reactor performing carbon, nitrogen, and phosphorus removal from synthetic wastewater. We found that FNA had a much stronger inhibitory effect on phosphorus (P) uptake and glycogen production than on poly-beta-hydroxyalkanoate degradation and nitrite reduction. The intracellular adenosine triphosphate levels decreased sharply during the FNA incubation, and the decreasing rates were positively correlated with increasing FNA concentrations. The electron transport activity of DPAOs when exposed to FNA displayed a similar trend. Further, at FNA concentrations above 0.044 mg HNO2-N/L, the anaerobic metabolism of DPAOs was initiated despite of the presence of nitrite, as evidenced by the release of phosphorus and the consumption of glycogen. DPAO metabolism did not recover completely from FNA inhibition in the subsequent FNA-free environment. The recovery rate depended on the concentration of FNA applied in the previous anoxic period. These results suggest that the inhibitory effects are diverse and may be attributable to different mechanisms operating simultaneously.