A pilot-scale horizontal subsurface flow constructed wetland (HFCW) was established to investigate the feasibility of removing nutrients from natural water in Lake Xijiu, Jiangsu Province, China. The HFCW, planted with reeds (Phragmites communis) and irises (Iris tectorum Maxim), was operated for 227 days. The mean removal efficiency of NH₄⁺-N was 36.7%, of total nitrogen (TN) 70.2%, of total phosphorus 75.3%, of permanganate index (COD_Mn) 35.8%, and of Chlorophyll a 70.0%. The effluent concentrations of these pollutants were all within the Grade III threshold of China’s water quality standards for surface water. Correlation analysis showed that TN removal was related to the COD_Mn concentration in the influent (r² = 0.65) and was closely related to the temperature (r² = 0.91). The HFCW had a water production efficiency of 98%–99% over the entire operational period, which suggests that it did not block easily. Results from 16S rDNA high-throughput sequencing showed that Proteobacteria (40.65%) dominated at the phylum level. At the genus level, the relative abundance of Nitrosomonadaceae (3.57%) was highest in autumn and the spatial distribution from the front to the back varied slightly from 2.57% to 2.99%. Our results will help researchers to develop optimal designs for HFCW systems to treat slightly polluted water.