Excessive fertiliser has been commonly applied in the soybean (Glycine max (L.) Merr.) cropping system in fertile Mollisols in Northeast China. However, it is necessary to understand how reducing nitrogen (N) fertiliser application may affect plant N acquisition and remobilisation, which is associated with photosynthetic carbon (C) assimilation and seed yield. The aim of this study was to investigate the origin of plant N (i.e. derived from N2 fixation, fertiliser or soil) under two different levels of N application, and the subsequent influence on C assimilation. A pot experiment was conducted with soybean grown in a Mollisol supplied with 5 mg N kg–1 soil (N5) or 100 mg N kg–1 soil (N100). Nitrogen was applied as 19.83% of 15N atom-excess in urea before sowing, and 13CO2 labelling was performed at the R5 (initial seed-filling) stage. Plants were harvested at R5 and full maturity stages to determine the 15N and 13C abundance in plant tissues. Seed yield and N content were not affected by different N rates. Symbiotically fixed N accounted for 64% of seed N in treatment N5, whereas fertiliser-derived N dominated seed N in N100, resulting in 58% of seed N. The proportion of soil-derived N in shoot and seed showed no difference between the two N treatments. A similar trend was observed for whole-plant N. The enhanced N2 fixation in N5 significantly increased assimilation of N and C during the seed-filling period compared with N100. Nodule density (nodule number per unit root length) and amount of photosynthetically fixed 13C in roots in N5 were greater than in N100. These results indicate that a greater contribution of N2 fixation to N assimilation during the seed-filling period is likely to meet N demand for maintaining soybean yield when fertiliser N supply is reduced. Greater allocation of photosynthetic C to roots and enhanced nodulation would greatly contribute to the alteration of N acquisition pattern under such condition.