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The specific mechanisms by which nitrogen affects the nodulation and nitrogen fixation of soybean plants are unclear. Investigating the relationship between nitrogen, nodulation and nitrogen fixation can provide new insights for the rational and proper use of nitrogen fertilizer in soybean plants. In this study, we grafted soybean roots to construct a dual-root system with a single nodulated side. Experiment I was performed at the third trifoliate leaf to initial seed filling (V3-R3) growth stages (for 30 days) for long-term nitrogen supply, and Experiment II was performed at the third trifoliate leaf to fourth trifoliate leaf (V3-V4) growth stages (for 5 days) for short-term nitrogen supply. For the two experiments, a nutrient solution providing ¹⁵NH₄¹⁵NO₃ or NH₄NO₃ as the nitrogen source was added to the non-nodulated side, while a nitrogen-free nutrient solution was added to the nodulated side. The concentrations of nitrogen supplied were 0 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L. The results showed the following: (1) Short-term nitrogen supply systematically regulated the specific nitrogenase activity (SNA), thereby inhibiting the acetylene reduction activity (ARA). Under long-term nitrogen supply, the recovery of SNA was generally consistent across treatments, and the concentration of nitrogen supplied systematically regulated the growth of root nodules, thereby inhibiting the ARA. (2) Using the ¹⁵N tracer method, the concentration of fertilizer nitrogen was positively correlated with the amount of nitrogen redistributed to other organs. Although the percentage of nitrogen derived from the atmosphere (Ndfa%) decreased significantly with increasing concentrations of nitrogen supplied, the effect on the accumulation of nitrogen fixed by nodules (N_accumulation of nodules) was not significant. By establishing the relationships between the ARA (measured by the acetylene reduction method), Ndfa% (based on ¹⁵N calculations), and N_accumulation nodules (based on ¹⁵N calculations), it was found that the ARA reliably reflected the Ndfa% but not the N_accumulation of nodules.