The study aims to evaluate the difference of nitrogen (N) utilization in peanut varieties with different nodulation efficiency and the contribution of different N sources to yield formation. Based on an outdoor pot experiment, N-15 isotope-labeled urea was used as a N source to investigate the effects of different N fertilization levels (N rates with 45, 75, 105, 135, 165 kg N ha(-1), defined as N45, N75, N105, N135 and N165 in the study, respectively) on peanut photosynthesis, photosynthate accumulation, yield, and N distribution and transport. The results showed that N application can improve peanut yield by creating photosynthesis, dry matter weight, and N accumulation, and the N105 treatment had the most significant effect. However, higher N applications inhibited the number of peanut root nodules. The ratio of N supply for peanuts from nodules, soil, and fertilizer at the pod setting stage was about 5:3:2, and the ratio of fertilizer distribution for low nodulation peanut variety of reproductive organ (pod) to nutrient organs (root, stem, leaf) was about 3:2, while the high nodulation variety was about 1:1. Biological N fixation is an important N source during peanut growth and development. Appropriate N fertilizer can further promote peanut growth and yield formation without inhibiting nodulation and N fixation. In agricultural production, optimizing N fertilizer management (105 similar to 135 kg N ha(-1)) in combination with using nodulation efficient peanut varieties not only promotes the N-cycling in agriculture, but also effectively reduce the waste of N fertilizer as well as environmental damage.