Two enhanced biological phosphorus removal (EBPR) sludges were used to investigate the effect of nitrite on anaerobic metabolism of phosphate accumulating organisms (PAOs). It is found that tolerance of PAOs to nitrite in two sludges were different. In the sludge with enriched PAOs cultures treating synthetic wastewater, when nitrite is above 10 mg/L, it inhibits VFA-uptake of PAOs, leading to decrease of PHA synthesis and increase of phosphate release. In the sludge with nitrogen and phosphorus removal by shortcut nitrification treating real domestic wastewater, anaerobic metabolism of PAOs is not inhibited even when nitrite is 30 mg/L; however, the presence of nitrite in anaerobic period causes denitrifying bacteria compete for limited carbon sources with PAOs, resulting in decrease of PHA synthesis and phosphate release. For the enriched PAOs cultures, the ratio of P/VFA obviously increases when nitrite is added in the reactor, indicating increased energy requirement for PAOs to take up VFA. The quantification results for PAOs in two sludges using fluorescence in-situ hybridization (FISH) show that the PAOs level is 55% in the enriched PAOs cultures, and 7.6% in the sludge with nitrogen and phosphorus removal by shortcut nitrification.