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Perennial grain crops intercropped with legumes are expected to use nitrogen (N) resources efficiently. A pot experiment using the 15N isotope dilution method demonstrated interspecific competition and use of N from the soil and N2 fixation in intermediate wheatgrass (Thinopyrum intermedium (Host) Barkworth & D.R. Dewey, IWG) and white clover (Trifolium repens L., WC) intercrops at five species-relative frequencies and four levels of inorganic N fertilizer in a replacement series design. The proportion of N in WC derived from the atmosphere increased from 39.7% in a sole crop to 70.9% when intercropped with IWG, and 10.1% N in IWG transferred from WC. Intermediate wheatgrass showed high fitness with maintained high total dry matter production at low relative frequencies. Decreasing IWG-relative frequency only increased dry matter and N accumulation of WC, resulting in increased amounts of N2 fixed. Increased levels of N fertilization increased the proportion of N acquired from the fertilizer in IWG and WC but decreased the N fixed by WC and N absorbed by IWG from the soil. Our study indicates that WC supply sufficient fixed N2 for IWG intercrop biomass yields under appropriate levels of soil N fertility and species-relative frequencies.