Among　the　earliest　synthetic　antipyretic　drugs,　phenacetin　(PNCT)　could　be　used　as　the　novel　partial　nitrification　(PN)　inhibitor　to　effectively　inhibit　nitrite-oxidizing　bacteria　(NOB).　In　practical　application,　the　rapidly　starting　of　PN　could　provide　stable　source　of　nitrite　for　anaerobic　ammonium　oxidation　(anammox)　process.　However,　impact　of　PNCT　on　anaerobic　ammonia　oxidizing　bacteria　(AnAOB)　and　its　underlying　mechanisms　were　not　clear.　In　this　research,　totally　14　times　of　PNCT　aerobic　soaking　treatment　were　performed　in　the　AnAOB　naturally　enrichment　system　to　improve　total　inorganic　nitrogen　removal　efficiency　(TINRE).　After　once　of　PNCT　treatment,　TINRE　rose　from　61.89　%　to　79.93　%.　After　14　times　of　PNCT　treatment,　NOB　Nitrospira　relative　abundance　decreased　from　9.82　%　to　0.71　%,　though　Candidatus　Brocadia　relative　abundance　also　declined,　it　might　gradually　adjust　to　PNCT　by　converting　the　leading　oligotype　species.　The　activity　and　relative　abundances　of　NOB　were　reduced　by　PNCT　via　decreasing　the　abundances　of　genes　amoA　and　nxrB,　enzymes　NxrA　and　NxrB.　Moreover,　Candidatus　Jettenia　and　Ca.　Brocadia　might　be　the　potential　host　of　qacH-01　and　they　played　the　crucial　role　in　the　shaping　profile　of　antibiotic　resistance　genes　(ARGs).　The　explosive　propagation　or　transmission　of　ARGs　might　not　take　place　after　PNCT　treatment.　©　2024　Elsevier　B.V.