The vertical-flow constructed wetland (VFCW) is a promising engineering technique for removal of excess nutrients and certain pollutants from wastewaters. This study investigated the uptake and removal of total nitrogen (TN) by Cyperus alternifolius from domestic wastewaters in simulated VFCWs. A total of eight simulated VFCW treatments, including two different substrates, two different wet-to-dry ratios, and with and without C. alternifolius species (2 × 2 × 2 = 8), were employed for an operation period of 2 years in this study. Results show that more TN was removed from the influent with the presence of C. alternifolius compared to without this plant species. A linear correlation existed between the aboveground biomass and its TN content. An increase in total biomass by 100 g resulted in an increase in TN accumulation in the aboveground biomass by 2.4 g. Large amounts of TN were removed by the aboveground biomass compared to those by substrate adsorption. Results suggest that C. alternifolius played a discernable role in removal of TN from wastewaters in the simulated VFCWs.