This　study　elucidated　the　differential　behaviors　of　hydrazine　(N2H4),　an　essential　intermediate　during　anaerobic　ammonia　oxidation　(anammox)　and　an　exogenous　stimulant　for　anammox,　in　sulfur-oxidizing　bacteria　(SOB)-mediated　sequential　denitrification　processes,　with　particular　relevance　to　systems　with　the　cooccurrence　or　synergy　of　SOB　and　anammox　bacteria.　Through　systematic　experiments,　we　revealed　that　SOB　lacked　the　capacity　to　directly　metabolize　N2H4　at　levels　of　≤　5　mg-N/L　which　did　not　affect　SOB　activity.　However,　a　previously　unrecognized　process-dependent　dichotomy　in　N2H4　transformation　was　discovered.　N2H4　remained　stable　during　NO2−　reduction.　By　contrast,　during　NO3−　reduction,　increasing　the　exogenous　N2H4　concentration　from　2　to　5　mg　N/L　elevated　N2H4　consumption　from　0.57　±　0.14　to　1.40　±　0.19　mg　N/L,　with　N2　being　the　main　product.　AlphaFold　3　prediction-based　molecular　docking　analyses　proved　that　this　divergence　originated　from　distinct　binding　capabilities　of　nitrate　reductase　(NAR)　and　nitrite　reductase　(NIR)　to　N2H4　during　denitrification,　suggesting　specific　NAR-mediated　cometabolism　in　SOB　(i.e.,　the　enzyme　concurrently　transforms　N2H4　while　acting　on　its　primary　substrate,　NO3−).　These　findings　advanced　the　fundamental　understanding　of　SOB-anammox　interactions.　Furthermore,　they　provided　critical　perspectives　for　regulating　engineered　water　treatment　systems　that　couple　sulfur-driven　autotrophic　denitrification　with　anammox.　©　2025　Elsevier　B.V.