Horizontal subsurface flow constructed wetlands (HSCWs) are widely use for wastewater treatment. The objective of this study is to assess the effects of substrate‐size selection and layout optimization on pollutant removal and microbial‐community‐distribution responses in HSCWs. Three pilot‐scale constructed wetlands (CWs) are established at Guilin University of Technology, China, to treat campus sewage. The three CWs include monolayer (CW1), three‐layer (CW2), and six‐layer (CW3) substrate structures with the hydraulic conductivity of the substrate increasing from the surface to the bottom in the multilayer CWs. Under an aerial influent‐loading rate of 0.38 m per day (volumetric loading rate of 0.63 per day), CW3 exhibits the highest removal performance for chemical oxygen demand (COD), NH4+‐N, and total nitrogen (TN), with mean values of 81, 81, and 74%, respectively, followed by CW2 (68, 71, and 60%, respectively) and CW1 (56, 46, and 41%, respectively). Nitrification is demonstrates to be the limiting factor of TN removal, and higher TN‐removal performance in the multilayer CWs is attributed to the higher proportions of nitrifiers, including ammonia‐oxidising bacteria (AOB) and nitrite‐oxidizing bacteria (NOB). Moreover, 454‐pyrosequencing shows a significantly different spatial distribution of the N‐transforming microbial community in multilayer HSCWs with substrate layout optimization.