Phenacetin　(PNCT)　belongs　to　one　of　the　earliest　synthetic　antipyretics.　However,　impact　of　PNCT　on　nitrifying　microorganisms　in　wastewater　treatment　plants　and　its　potential　microbial　mechanism　was　still　unclear.　In　this　study,　PN　could　be　initiated　within　six　days　by　PNCT　anaerobic　soaking　treatment　(8　mg/L).　In　order　to　improve　the　stable　performance　of　PN,　21　times　of　PNCT　aerobic　soaking　treatment　every　three　days　was　conducted　and　PN　was　stabilized　for　191　days.　After　PN　was　damaged,　ten　times　of　PNCT　aerobic　soaking　treatment　every　three　days　was　conducted　and　PN　was　recovered　after　once　soaking,　maintained　over　88　days.　Ammonia　oxidizing　bacteria　might　change　the　dominant　oligotype　to　gradually　adjust　to　PNCT,　and　the　increase　of　abundance　and　activity　of　Nitrosomonas　promoted　the　initiation　of　PN.　For　nitrite-oxidizing　bacteria　(NOB),　the　increase　of　Candidatus　Nitrotoga　and　Nitrospira　destroyed　PN,　but　PN　could　be　recovered　after　once　aerobic　soaking　illustrating　NOB　was　not　resistant　to　PNCT.　KEGG　and　COG　analysis　suggested　PNCT　might　disrupt　rTCA　cycle　of　Nitrospira,　resulting　in　the　decrease　of　relative　abundance　of　Nitrospira.　Moreover,　PNCT　did　not　lead　to　the　sharp　increase　of　absolute　abundances　of　antibiotic　resistance　genes　(ARGs),　and　the　risk　of　ARGs　transmission　was　negligible.　©　2024　Elsevier　B.V.