Bioretention system has been commonly recognized as an emerging management method to control urban rainwater runoff. However, nitrogen conversion mechanisms in bioretention system in response to formulated soil medium remains unknown. In this work, five bioretention columns with different formulated soil medium were established, and results indicated that all bioretention columns achieved high NH4+-N removal rate, but exhibited significantly different NO3--N removal performance. High-throughput sequencing results suggested that nitrification might be achieved by heterotrophic nitrifying bacteria, while Pseudomonas and Rubellimicrobium may be the main denitrifying bacteria, which served as anaerobic and aerobic denitrifier, respectively. Heterotrophic nitrification-anaerobic/aerobic denitrification may the microbial pathways to realize effective nitrogen removal performance in these bioretention systems. Bioretention system with moderate silt content (NC, 10%) displayed higher abundant potential denitrifier, leading to better nitrogen removal performance. Besides, soil adsorption played an important role in nitrogen removal, particularly when activated carbon was added. This work could provide guidance of formulated soil medium optimization for the application of bioretention systems.