Natural estrogens, including estrone (E1), 17β-estradiol (E2), and estriol (E3), are potentially carcinogenic pollutants commonly found in water and soil environments. Bacterial metabolic pathway of E2 has been studied; however, the catabolic products of E3 have not been discovered thus far. In this study, Novosphingobium sp. ES2-1 was used as the target strain to investigate its catabolic pathway of E3. The metabolites of E3 were identified by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) combined with stable 13C3-labeling. Strain ES2-1 could almost completely degrade 20 mg∙L-1 of E3 within 72 h under the optimal conditions of 30°C and pH 7.0. When inoculated with strain ES2-1, E3 was initially converted to E1 and then to 4-hydroxyestrone (4-OH-E1), which was then cleaved to HIP (metabolite A6) via the 4, 5-seco pathway or cleaved to the B loop via the 9,10-seco pathway to produce metabolite with a long-chain ketone structure (metabolite B4). Although the ring-opening sequence of the above two metabolic pathways was different, the metabolism of E3 was achieved especially through continuous oxidation reactions. This study reveals that, E3 could be firstly converted to E1 and then to 4-OH-E1, and finally degraded into small molecule metabolites through two alternative pathways, thereby reducing E3 pollution in water and soil environments.