Much nitrogen (N) is lost in high-input protected cropping systems mainly via leaching of not only nitrate-N but also extractable organic N (EON), but the role of EON in this process is poorly appreciated. A consecutive 3-year plot experiment was conducted to investigate the impact of co-application of manures with chemical N fertilizer on N accumulation and loss in a greenhouse soil rotationally planted with cucumber or tomato and lettuce. Application of manures significantly enhanced the average contents and stocks of NO3 −-N, EON, and total N (TN) in 0- to 60-cm soil layer, although EON accumulated within growing season, while NO3 −-N accumulated with fluctuation, and TN accumulated gradually throughout the 3-year experiment. With application rate at 120 or 180 t dry manures per hectare per 3 years, the corresponding apparent N surplus was 2710 or 3924 kg⋅ha−1 per 3 years. Due to little increase of biomass N uptakes during vegetable seasons, the accumulated N in soil profile would be a potential loss source, largely via leaching of both nitrate and EON. Application of manures slowed soil acidification but intensified secondary salinization of the greenhouse soil. Considering the manures-induced high soil N accumulation and loss, well-balanced evaluation of the role of manures in high-input agricultural ecosystems is needed.