Background and Aims Knowing the impact of cultural practices on nitrogen (N) dynamics in perennial crops is critical to promote N use efficiency. This study focused on the impact of crop regulation on the plant N dynamics, on the fruit N composition, and on the N fertilisation use efficiency. Methods and Results A large crop load gradient was set in a homogeneous plot of the grape cultivar Chasselas. Fertilisation in the form of N-15-labelled foliar urea allowed the measurement of N uptake and partitioning among plant fractions. Dry mass, carbon, and N dynamics were assessed over two consecutive seasons. Crop regulation did not affect grape N concentration at harvest. Both N uptake and root N mobilisation were reduced in response to crop regulation. Fertilisation efficiency was higher under high-yield conditions in terms of N uptake and grape N accumulation. The carry-over effects of crop regulation in the following year were highlighted. Conclusions Crop regulation strongly affects the overall plant N cycle, that is, uptake, distribution and release. Crop regulation improves must sugar concentration at harvest, while N concentration remained unchanged. The efficiency of N fertilisation varies greatly with crop load, which limits the interest of fertilisation under low-yield conditions. Significance of the Study These results contribute to the development of accurate nutrition models and sustainable cultural practices.