The anaerobic-intermittently aerobic (AIA) process was operated for the enhanced biological phosphorus and nitrogen removal over 2 years. A bench-scale AIA reactor operated in a continuous- flow anaerobic and alternating anoxic-aerobic mode was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under the anaerobic zone, carbon source was taken up, polyhydroxyalkanoates (PHAs) were formed, and accomplished phosphorus release. The simultaneous phosphate uptake and denitrification by the denitrifying phosphate accumulating organisms (DePAOs) was observed even though the PHAs in cells were oxidized in the aerobic phase before the anoxic phase. Ammonium was oxidized to nitrate under the aerobic phase, and nitrate was reduced to nitrogen gas under the anoxic phase. As the nitrate concentration increased, phosphate uptake rate and denitrification rate decreased, whereas the release of phosphate was accelerated with the addition of the external carbon source. The secondary phosphate release was not associated with the nitrate concentration, but was significantly related to the remained PHA concentration. The fraction of DePAOs among the entire phosphate accumulating organisms (PAOs) population was about 60% in the AIA reactor. Results from the fluorescent in situ hybridization confirmed that Rhodocyclus sp. was abundant in the AIA process, and Rhodocyclus-like organisms played major roles in the simultaneous phosphate uptake and denitrification under the anoxic condition, indicating the phosphate uptake under the anoxic phase was relatively slower than that under the aerobic phase.