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Thermal activation of persulfate was confirmed to be effective in the destruction of organic pollutants. Microwave heating has different inherent mechanism from that of conventional heating, and the application of microwave heating to chemical reactions has attracted great interest. The objective of this study was to evaluate the degradation of sulfamethoxazole (SMX) in a microwave-activated persulfate (MW/PS) system. The results indicated that MW/PS degradation of SMX followed pseudo-first-order kinetics, and compared with conventional heating, microwave heating has a special effect on SMX degradation with higher reaction rate and shorter process time. The process of SMX degradation was accelerated by higher reaction temperature, persulfate dose or pH in the MW/PS system, while higher initial SMX concentration and the presence of phosphates slowed down the degradation rates. High level of chloride showed some inhibition on the SMX degradation, while low chloride level and carbonate enhanced the SMX degradation. 3-Amino-5-methylisoxazole, sulfanilic acid, hydroxyl-SMX and nitroso-SMX derivatives were identified as the major degradation intermediate products by HPLC/MS. The possible reaction pathways including hydroxylation of the benzene ring, oxidation of the amine group at the benzene ring and the S–N cleavage were proposed. The acute toxicity tests with Photobacterium phosphoreum, Vibrio fischeri and Vibrio qinghaiensis indicated that the inhibitory effect of the 10-time diluted unheated SMX mixture solution being 22.6–48.0%, increased to >99.9% after 4 min and decreased to