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Bioremediation has been used as an environmentally-friendly, energy-saving and efficient method for removing pollutants. However, there have been very few studies focusing on the specific antibiotic-degrading microorganisms in the activated sludge and their degradation mechanism. Two strains of cefalexin-degrading bacteria (Rhizobium sp. (CLX-2) and Klebsiella sp. (CLX-3)) were isolated from the activated sludge in this study. They were capable of rapidly eliminating over 99% of cefalexin at an initial concentration of 10 mg l−1 within 12 h. The exponential phase of cefalexin degradation happened a little earlier than that of bacterial growth. The first-order kinetic model could elucidate the biodegradation process of cefalexin. The optimized environmental temperature and pH values for rapid biodegradation by these two strains were found to be 30°C and 6.5–7, respectively. Furthermore, two major biodegradation metabolites of CLX-3, 7-amino-3-cephem-4-carboxylic acid and 2-hydroxy-3-phenyl pyrazine were identified using UHPLC-MS and the biodegradation pathway of cefalexin was proposed. Overall, the results showed that Rhizobium sp. (CLX-2) and Klebsiella sp. (CLX-3) could possibly be useful resources for antibiotic pollution remediation.