Maintaining seed yield under low N inputs is a major issue for breeding, which requires thoroughly exploiting the genetic diversity of processes related to Nitrogen Use Efficiency (NUE). However, dynamic analysis of processes underlying genotypic variations in NUE in response to N availability from sowing to harvest are scarce, particularly at the whole-plant scale. This study aimed to dynamically decipher the contributions of Nitrogen Uptake Efficiency (NUpE) and Nitrogen Utilization Efficiency (NUtE) to NUE and to identify traits underlying NUpE genetic variability throughout the growth cycle of rapeseed. Three experiments were conducted under field-like conditions to evaluate seven genotypes under two N conditions. We developed NUE_DM (ratio of total plant biomass to the amount of N available) as a new proxy of NUE at harvest, valid to discriminate genotypes from the end of inflorescence emergence, and N conditions as early as the beginning of stem elongation. During autumn growth, NUpE explained up to 100% of variations in NUE_DM, validating the major role of NUpE in NUE shaping. During this period, under low N conditions, up to 53% of the plant nitrogen was absorbed and NUpE genetic variability resulted not from differences in Specific N Uptake but in fine-root growth. NUtE mainly contributed to NUE_DM genotypic variation during the reproductive phase under high-N conditions, but NUpE contribution still accounted for 50–75% after flowering. Our study highlights for the first time NUpE and fine-root growth as important processes to optimize NUE, which opens new prospects for breeding.