As an important oil crop in China, rapeseed(Brassica napus L.) has a high demand for nitrogen(N) and the N deficiency in soil is an important limiting factor for rapeseed production. Taking Brassica napus L. as the experimental material, low N(LN, 0.3 mmol/L NO3–) and high N(HN, 6.0 mmol/L NO3–) treatments were set up in this study. The results showed that the low N promoted root elongation of rapeseed, and induced higher root/shoot ratio and N physiological efficiency. In the meantime, low N increased cytokinin content in the elongation zone and auxin content in the mature zone of rapeseed root tip, thereby promoted cell division in the elongation zone and cell elongation in the mature zone. Low N treatment decreased pectin and hemicellulose content in cell walls of rapeseed roots, while had no significantly effects on hemicellulose and lignin. Transcriptome results showed that the increase of pectin content was mainly due to the inhibition on expression of pectin degrading enzyme related genes by low N, and the effect on cellulose content was attributed to the increase of cellulose synthesis and the decrease of cellulose degradation. At the same time, the up-regulated expression of genes related to expansin also showed that low N promoted the relaxation of cell walls and was conducive to cell expansion and division in roots. Conclusively, the study revealed the mechanism underlying root elongation affected by alterations on cell wall structure and components under N deficiency.