Sulfonamides　are　frequently　detected　with　high　concentrations　in　various　environments　and　was　regarded　as　a　serious　environmental　risk　by　fostering　the　dissemination　of　antibiotic　resistance　genes.　This　study　for　the　first　time　reported　a　strain　SNF1　affiliated　with　Hydrogenophaga　can　efficiently　degrade　sulfamethoxazole　(SMX).　Strain　SNF1　prefers　growing　under　extra　carbon　sources　and　neutral　condition,　and　could　degrade　500　mg/L　SMX　completely　within　16　h.　Under　the　conditions　optimized　by　response　surface　method　(3.11　g/L　NaAc,　0.77　g/L　(NH4)2SO4,　pH　=　7.53,　and　T　=　34.38　℃),　a　high　removal　rate　constant　0.5104　/h　for　50　mg/L　SMX　was　achieved.　Coupling　the　intermediate　products　identification　with　comparative　genomic　analysis,　a　novel　SMX　degradation　pathway　was　proposed.　Unlike　Actinomycetota　degraders,　SMX　was　deaminized　and　ring　ortho-hydroxylated　in　strain　SNF1　using　a　Rieske　dioxygenase　in　combination　with　glutamine　synthetase　system.　Rieske　dioxygenase　gene　expression　was　up-regulated　by　1.09　to　6.02-fold　in　response　to　100　mg/L　SMX.　When　SMX　is　fully　degraded,　its　antimicrobial　activity　drops　by　over　90　%,　and　its　anticipated　toxicity　to　aquatic　organisms　were　overall　reduced.　These　findings　provided　new　insights　into　SMX-degrading　microorganisms　and　mechanisms　and　highlighted　the　potential　of　Hydrogenophaga.　sp.　SNF1　for　biological　elimination　of　SMX　from　wastewater.　©　2024　Elsevier　B.V.