Organophosphate esters (OPEs) are emerging contaminants of growing concern, and there is limited information about the bacterial transformation of OPEs. In this study, we investigated the biotransformation of tris(2-butoxyethyl) phosphate (TBOEP), a frequently detected alkyl-OPE by a bacterial enrichment culture under aerobic conditions. The enrichment culture degraded 5 mg/L TBOEP following the first-order kinetics with a reaction rate constant of 0.314 h-1. TBOEP was mainly degraded via ether bond cleavage, evidenced by the production of bis(2-butoxyethyl) hydroxyethyl phosphate, 2-butoxyethyl bis(2-hydroxyethyl) phosphate, and 2-butoxyethyl (2-hydroxyethyl) hydrogen phosphate. Other transformation pathways include terminal oxidation of the butoxyethyl group and phosphoester bond hydrolysis. Metagenomic sequencing generated 14 metagenome-assembled genomes (MAGs), showing that the enrichment culture primarily consisted of Gammaproteobacteria, Bacteroidota, Myxococcota, and Actinobacteriota. One MAG assigned to Rhodocuccus ruber strain C1 was the most active in the community, showing upregulation of various monooxygenase, dehydrogenase, and phosphoesterase genes throughout the degradation process, and thus was identified as the key degrader of TBOEP and the metabolites. Another MAG affiliated with Ottowia mainly contributed to TBOEP hydroxylation. Our results provided a comprehensive understanding of the bacterial TBOEP degradation at community level.