Plant development greatly influences the composition structure and functions of microbial community in constructed wetlands (CWs) via plant root activities. However, our knowledge of the effect of plant development on microbial nitrogen (N) cycle is poorly understood. Here, we investigated the N removal performance and microbial structure in subsurface flow CWs at three time points corresponding to distinct stages of plant development: seedling, mature and wilting. Overall, the water parameters were profoundly affected by plant development with the increased root activities including radial oxygen loss (ROL) and root exudates (REs). The removal efficiency of NH4+-N was significantly highest at the mature stage (p < 0.01), while the removal performance of NO3−-N at the seedling stage. The highest relative abundances of nitrification- and anammox-related microbes (Nitrospira, Nitrosomonas, and Candidatus Brocadia, etc.) and functional genes (Amo, Hdh, and Hzs) were observed in CWs at the mature stage, which can be attributed to the enhanced intensity of ROL, creating micro-habitat with high DO concentration. On the other hand, the highest relative abundances of denitrification- and DNRA-related microbes (Petrimonas, Geobacter, and Pseudomonas, etc.) and functional genes (Nxr, Nir, and Nar, etc.) were observed in CWs at the seedling and wilting stages, which can be explained by the absence of ROL and biological denitrification inhibitor derived from REs. Results give insights into microbial N cycle in CWs with different stages of plant development. More importantly, a potential solution for intensified N removal via the combination of practical operation and natural regulation is proposed.