Abstract: As one of the most critical environmental factors driving plant growth, soil N availability significantly influences the composition and quantity of root exudates. Concerning the environmental benefits, the inputs reduction of N fertilizer can be an attractive option for crop production in a more sustainable agriculture system. However, the pattern of root exudates in response to the reduced N levels remains poorly understood, especially for maize (Zea mays L.) plants. Metabolomics of root exudates can potentially help us to better understand the chemical communication between roots, soils and organisms in the rhizosphere. In this study, to characterize root exudation pattern of maize plants grown under conditions with reduced N fertilizer at metabolomics level, three methods were developed for collecting root exudates from maize seedling planted in soils through soil leachate (SL), rhizosheath soil extraction (RS) and distilled water cultivation (DWC), respectively. The metabolomics of root exudates collected by different methods under nitrogen fertilizer reduction conditions were investigated based on ¹H NMR spectroscopy. Partial least squares projection to latent structures-discriminant analysis was performed to quantify the difference of metabolomics among samples and conditions. The results showed that the whole profilings of ¹H NMR were distinctly different among root exudates obtained by three methods, and the major compounds contributing to the discrimination also varied. The most important exudates that differentiated the samples between SL and RS methods were glucose and alanine while more exudates such as acetate, lactate, succinate, sucrose, alanine, leucine, isoleucine and valine accounted for the discrimination between SL and DWC methods. In addition, the most important exudates that differentiated the samples between RS and DWC methods were acetate, lactate, succinate and isoleucine. The peak signals of ¹H NMR and number of assigned metabolites in root exudates detected by method of SL were less while that were rather more in the methods of RS and DWC; and sugars, organic acids and amino acids in root exudates of maize seedlings were detected by the latter two methods. In contrast to the control of 100%-N level (corresponding to the conventional application rate of 180 kg·hm^-2), the levels of α-glucose, malate, leucine and valine increased significantly in root exudates of maize seedlings in treatment with 85%-N level. However, the level of exudates decreased with reducing fertilizer N application rate and its difference between control and 55%-N treatment was not significant. The changes of root exudates may influence soil organic matter turnover and lead to an increase in plant-available N. Our results indicated that maize seedlings might adapt to the variation of nitrogen nutrient situation in soil by regulation of exudation. Metabolomics analysis of root exudates based on ¹H NMR spectroscopy combined with sample collecting methods of rhizosheath soil extraction and distilled water cultivation could provide important theoretical basis for the study on the rhizosphere ecology and nitrogen nutrition. The possible ecological roles of root exudates in response to N reduction should be fully elucidated in the future. A combined approach involving different metabolomic tools will facilitate the understanding of belowground chemical communications and rhizosphere interactions under conditions of N reduction.