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 500mg/L SMX completely within 16h. Under the conditions optimized by response surface method (3.11g/L NaAc, 0.77g/L (NH4)2SO4, pH = 7.53, and T = 34.38℃), a high removal rate constant 0.5104 /h for 50mg/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 100mg/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.