Oxytetracycline (OTC) is widely used in mariculture as a prophylactic and feed supplement, leading to its frequent detection at high concentrations in coastal environments and posing risks to marine ecosystems and human health. To address this issue, a novel OTC-degrading strain, Brucella sp. F-6, was isolated from estuarine sediments. It was first verified to degrade OTC into small molecules with a degradation rate of 92.06±0.98% whitin 48 h. The OTC degradation pathway was explored, and Glycine betaine monooxygenase (Gbs) and sarcosine oxidase (Sox) were proposed as candidate degradation proteins based on transcriptomic analysis and degradation product identification. The genes encoding candidate degradation protein Gbs were significantly upregulated even at an environmentally relevant concentration of 0.1 μg/L OTC. The Cu-OTC chelate was suggested to be involved in OTC detoxification. Strain F-6 resisted OTC through multiple mechanisms, including drug efflux, outer membrane protection, ribosomal protection, and DNA repair. Meanwhile, nitrogen metabolism, one-carbon metabolism, and the antioxidant system were affected or inhibited. However, only drug efflux was activated under environmental concentrations of OTC. These findings have important implications for research on antibiotic removal and environmental toxicity assessment.