Berberine (BBR) is widely used as a botanical pesticide due to its broad-spectrum antibacterial and antifungal activities. However, BBR degradation pathway in soil microorganisms, which determines its impact on soil environment, remains poorly understood. Herein, a novel BBR-degrading bacterium Agrobacterium sp. V1 was isolated and characterized. Agrobacterium sp. V1 was able to utilize BBR as the sole carbon source for cell growth, and 50μg/mL of BBR was completely degraded within 48h. To reveal the possible BBR degradation pathway, whole genome sequencing of Agrobacterium sp. V1 was conducted, and proteins in Agrobacterium sp. V1 were aligned with enzymes involved in BBR biosynthesis in Rhizoma Coptidis. The results indicated that more than 60% of enzymes in BBR biosynthesis pathway had orthologs in Agrobacterium sp. V1. Combined with the primary mass spectra of BBR metabolites, a novel BBR degradation pathway in this bacterium was proposed. In summary, the proposed BBR degradation pathway offered new insights into the impact of BBR to the environment and also provided a reference for studying BBR metabolism in microorganisms.