Nickel (Ni(II)) is commonly present in municipal and industrial wastewaters, and thus its potential toxicity to activated sludge in wastewater treatment plants attracts increasing concerns. Although considerable efforts have been paid to this topic, the potential effect of Ni(II) on biological phosphorus removal has not been reported. In this work, short-term and long-term effects of Ni(II) in the range of 0.1–10mg·L−1 on enhanced biological phosphorus removal (EBPR) were therefore investigated. Compared with the control, short-term exposure to 1 and 10mg·L−1 of Ni(II) resulted in the decrease of phosphorus removal efficiency from 99.7% to 38.3% and 0, respectively. The phosphorus removal was unaffected after short-term exposure to 0.1mg·L−1 of Ni(II), but it was completely collapsed after 30-day exposure. The mechanism studies revealed that the cell membrane of microorganisms in activated sludge was not damaged, though the production of reactive oxygen species increased with the increase of Ni(II) exposure concentration. However, the presence of Ni(II) inhibited the anaerobic release of polyphosphate and the activity of exopolyphosphatase but enhanced the transformations of poly-3-hydroxyvalerate and glycogen. Microbial community investigation with high-throughput sequencing analysis showed that alphaproteobacterial glycogen accumulating organisms instead of polyphosphate accumulating organisms became the predominant microorganisms in EBPR systems after long-term exposure to Ni(II).