The soybean (Glycine max L. Merr.) is a crop with a high demand for nitrogen (N). The root nodules that form in soybeans can fix atmospheric N effectively, yet the goal of achieving high yields cannot be met by relying solely on nodule-fixed N. Nonetheless, the application of N fertilizer may inhibit nodule formation and biological N fixation (BNF), but the underpinning mechanisms are still unclear. In this study, we grafted the roots of non-nodulated soybeans onto nodulated soybeans to generate plants with dual root system. The experiment included three treatments conducted under sand culture conditions with NO 3 − and NH 4 + as N sources. Treatment I: The non-nodulated roots on one side received 50 mg·L−1 15 NO 3 − or 15NH4+, and the nodulated roots on the other side were not treated. Treatment II: The non-nodulated roots received 50 mg·L−1 15 NO 3 − or 15 NH 4 + , and the nodulated roots received 50 mg·L−1 14 NO 3 − or 14 NH 4 + . Treatment III: Both non-nodulated and nodulated roots received 50 mg·L−1 15 NO 3 − or 15 NH 4 + . The results showed the following: (1) Up to 81.5%–87.1% of the N absorbed by the soybean roots and fixed by the root nodules was allocated to shoot growth, leaving 12.9%–18.5% for root and nodule growth. Soybeans preferentially used fertilizer N in the presence of a NO 3 − or NH 4 + supply. After the absorbed fertilizer N and nodule-fixed N was transported to the shoots, a portion of it was redistributed to the roots and nodules. The N required for root growth was primarily derived from the NO 3 − or NH 4 + assimilated by the roots and the N fixed by the nodules, with a small portion translocated from the shoots. The N required for nodule growth was primarily contributed by nodule-fixed N with a small portion translocated from the shoots, whereas the NO 3 − or NH 4 + that was assimilated by the roots was not directly supplied to the nodules. (2) Based on observations of the shoots and one side of the roots and nodules in the dual root system as an N translocation system, we proposed a method for calculating the N translocation from soybean shoots to roots and nodules during the R1–R5 stages based on the difference in the 15N abundance. Our calculations showed that when adding N at a concentration of 50 mg·L−1, the N translocated from the shoots during the R1–R5 stages accounts for 29.6%–52.3% of the N accumulation in nodulated roots (Rootn) and 9.4%–16.6% of the N accumulation in Nodulen of soybeans. Through the study of this experiment, the absorption, distribution and redistribution characteristics of fertilizer N and root nodule N fixation in soybean can be clarified, providing a theoretical reference for analyzing the mechanisms of the interaction between fertilizer N and nodule-fixed N.