Nitrogen (N) is the main nutrient of plants, and low nitrogen usually affects plant growth and crop yield. The traditional Chinese herbal medicine Dendrobium officinale Kimura et. Migo is a typical low nitrogen-tolerant plant, and its mechanism in response to low nitrogen stress has not previously been reported. In this study, physiological measurements and RNA-Seq analysis were used to analyse the physiological changes and molecular responses of D. officinale under different nitrogen concentrations. The results showed that under low nitrogen levels, the growth, photosynthesis and superoxide dismutase activity were found to be significantly inhibited, while the activities of peroxidase and catalase, the content of polysaccharides and flavonoids significantly increased. Differentially expressed genes (DEGs) analysis showed that nitrogen and carbon metabolisms, transcriptional regulation, antioxidative stress, secondary metabolite synthesis and signal transduction all made a big difference in low nitrogen stress. Therefore, copious polysaccharide accumulation, efficient assimilation and recycling of nitrogen, as well as rich antioxidant components play critical roles. This study is helpful for understanding the response mechanism of D. officinale to low nitrogen levels, which might provide good guidance for practical production of high quality D. officinale .