Biological nutrient removal process is pivotal in controlling nitrogen and phosphorus from municipal wastewater treatment and reclamation plants in the water environment. The efficiency of municipal wastewater treatment is affected by the fluctuation of influent quality. In this study, the impact of N loading on the removal efficiency and the microbial community structure of activated sludge with long sludge retention time were investigated. The results showed that the effluent NH4+-N and NO2--N concentration were lower than 0.5 mg/L, and the TN removal efficiencies were 79.77% and 65.80%, respectively. The dehydrogenase activity, specific oxygen uptake rate, specific nitrification rate, and specific denitrification rate increased as the N loading increasing. At the phylum level, the most dominant bacterial was affiliated with Proteobacteria in the two systems, accounting for 60.65% and 48.62%, respectively. At the genus level, Acinetobacter, Thauera, and Zoogloea played significant roles in denitrification. The ammonia oxidation bacteria and nitrite-oxidizing bacteria were of the genus Nitrosomonas (r-strategist) and Nitrospira (K-strategist).