Background: Berberine (BBR) is a pharmaceutical chemical with a broad antibacterial spectrum, and its biological treatment has been of research and practical interest. In this study, a pure bacterial strain B16 was isolated from the activated sludge in a pharmaceutical wastewater treatment plant. The aim of the study is to characterize the properties of the strain B16, especially its BBR degradation capability. Methods: The identification of strain B16 was conducted by visual observation, as well as biochemical and phylogenetic analysis. The degradation kinetics of strain B16 was tentatively described by Haldane model. Results: The strain B16 was 100% determined as a Sphingopyxis sp. The kinetic parameters of BBR degradation by strain B16 were as follows: Vmax 54.73 ± 5.54 mg (g MLSS · h)−1, Km 66.68 ± 8.95 mg L−1, and Ki 43.16 ± 5.92 mg L−1, with an R2 of 0.996. Stain B16 exhibited considerable capability of BBR degradation. BBR of initial concentration 40 mg L−1 could be completely degraded in 48 h under optimal conditions. Conclusions: strain B16 was the first pure culture found with the ability to totally mineralize BBR, indicating the potential of B16 application in real industrial processes.