Nitrogen (N) deficiency is one of the pivotal environmental factors that induce leaf senescence. However, little is known regarding the impact of low N on root senescence in cotton. Thus, the objective of this study was to investigate the effect of low nitrogen on root senescence. In this study, the molecular mechanism of cotton root senescence in response to nitrogen deficiency was investigated by combing physiological and transcriptomic analysis when no nitrogen and normal nitrogen (138mg N·kg−1 soil). The results showed that: (1) nitrogen starvation induced the premature senescence of leaf, while delaying root senescence. (2) The increase in catalase (CAT) activity at 60, 80, and 100days after emergence (DAE), combined with decrease of malonaldehyde content at 60, 80, and 100 DAE, and the content of abscisic acid (ABA), all of these contributed to the delay of root senescence by low nitrogen treatment. (3) To study the molecular mechanisms underlying root senescence, the gene expression profiling between low nitrogen and normal nitrogen treatments were compared pairwise at 20, 40, 60, 80, and 100 DAE. A total of 14,607 genes were identified to be differentially expressed at these five points. (5) Most genes involved in glutathione (GSH) and ascorbate peroxidase (APX) synthesis were upregulated, while ABA, apoptosis, caspase, and cell cycle-related differentially expressed genes (DEGs) were downregulated. Coupled with the physiology data, these results provide new insights into the effect of nitrogen starvation on root senescence.