Appropriate　inhibitors　might　play　important　roles　in　achieving　ammonia　retention　in　biological　wastewater　treatment　and　its　reuse　in　agriculture.　In　this　study,　the　feasibility　of　epsilon-poly-l-lysine　(ϵ-PL)　as　a　novel　natural　ammonia　oxidation　inhibitor　was　investigated.　Significant　inhibition　(ammonia　oxidation　inhibition　rate　was　up　to　96.83%)　was　achieved　by　treating　the　sludge　with　ϵ-PL　(400　mg/L,　12　h　soaking)　only　once　and　maintaining　for　six　cycles.　Meanwhile,　the　organic　matter　and　nitrite　removal　was　not　affected.　This　method　was　effective　under　the　common　environmental　conditions　of　biological　wastewater　treatment.　Metatranscriptome　uncovered　the　possible　action　mechanisms　of　ϵ-PL.　The　ammonia　oxidation　inhibition　was　due　to　the　co-decrease　of　Nitrosomonas　abundance,　ammonia　oxidation　genes,　and　the　cellular　responses　of　Nitrosomonas.　Thauera　and　Dechloromonas　could　adapt　to　ϵ-PL　by　stimulating　stress　responses,　which　maintained　the　organic　matter　and　nitrite　removal.　Importantly,　ϵ-PL　did　not　cause　the　enhancement　of　antibiotic　resistance　genes　and　virulent　factors.　Therefore,　ϵ-PL　showed　a　great　potential　of　ammonia　retention,　which　could　be　applied　in　the　biological　treatment　of　wastewater　for　agricultural　reuse.　©　2023　American　Chemical　Society.