Abstract: Wetlands are ecotones between aquatic and terrestrial ecosystems, and play an important role in intercepting nitrogen sources on the land and reducing nitrogen levels in water bodies. Plants play a very important role in removing nitrogen in wetlands, and the plant harvesting (cutting) time affects the removing efficiency. During the plant wilting period (winter), there is inevitably a low nitrogen removal efficiency from wetlands. Understanding the optimal harvest time of reed (Phragmites australis) will allow the use of management practices to balance the trade-off between nitrogen removal and the sustainability of wetlands. An experiment was set up to investigate the nitrogen removal efficiency and root respiratory metabolism in reed wetlands for two years (discontinuity harvesting between years; the first year (from July 2017 to March 2018), influent TN 17.60-34.65 mg/L; the second year (from August 2018 to April 2019), influent TN 3.16-10.03 mg/L)). To identify the optimal harvest time: W1 (un-harvest), W2 (harvest before wilting), W3 (harvest at mid-wilting), and W4 (harvest at late wilting). The results showed that: (1) Harvesting decreased wetland nitrogen removal in both years, with later harvest time producing a smaller decrease in total nitrogen (TN) and ammonium (NH₄⁺-N) removal. (2) The root activity and hexokinase (HK), pyruvate kinase (PK) and phosphofructokinase (PFK) activities were significantly enhanced in winter when harvest before wilting in the year with higher influent TN concentration, and the wetland nitrogen removal was dominated by isocitric dehydrogenase (ICDH) and HK activities. While harvest time had little effect on root activity and HK, PK and PFK activities in the year with lower influent TN concentration, and HK activity had the greatest effect on wetland nitrogen removal. (3) The root ICDH and cytochrome oxidase (COX) activities decreased in both two-year experiments, but the effect of late wilting harvest was relatively small. The results show that there is considerable relationship between nitrogen removal in wetland and the characteristics of root respiration metabolism in winter, which is affected by the harvest time.