Cotton (Gossypium spp.) is the most important fibre crop, with desirable characteristics preferred for textile production. Cotton fibre output relies heavily on nitrate as the most important source of inorganic nitrogen (N). However, nitrogen dynamics in extreme environments limit plant growth and lead to yield loss and pollution. Therefore, nitrogen use efficiency (NUE), which involves the utilisation of the ‘right rate’, ‘right source’, ‘right time’, and ‘right place’ (4Rs), is key for efficient N management. Recent omics techniques have genetically improved NUE in crops. We herein highlight the mechanisms of N uptake and assimilation in the vegetative and reproductive branches of the cotton plant while considering the known and unknown regulatory factors. The phylogenetic relationships among N transporters in four Gossypium spp. have been reviewed. Further, the N regulatory genes that participate in xylem transport and phloem loading are also discussed. In addition, the functions of microRNAs and transcription factors in modulating the expression of target N regulatory genes are highlighted. Overall, this review provides a detailed perspective on the complex N regulatory mechanism in cotton, which would accelerate the research toward improving NUE in crops.