Transcriptome analysis revealed the potential mechanism of nitrogen regulating steviol glycosides synthesis via shifting of leaf carbon metabolic flux or inducing certain transcription factors. Nitrogen (N) plays key regulatory roles in both stevia (Stevia rebaudiana) growth and the synthesis of its functional metabolite steviol glycosides (SGs), but the mechanism by which this nutrient regulates SGs synthesis remains to be elucidated. To address this question, a pot experiment was performed in a greenhouse where stevia plants fertilized with N (the control as CK plants) and compared with plants without the supply of N. Physiological and biochemical analyses were conducted to test the growth and metabolic responses of plants to N regimes. Our results showed that N deficiency significantly inhibited plant growth and leaf photosynthesis, while increased leaf SGs contents in stevia (49.97, 46.64 and 84.80% respectively for rebaudioside A, stevioside, and rebaudioside C), which may be partly due to "concentration effect". Then, transcriptome analysis was conducted to understand the underlying mechanisms. A total of 535 differentially expressed genes were identified, and carbon metabolism-related events were highlighted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Many of these genes were significantly upregulated by N-deficiency, including those involved in "phenylpropanoid biosynthesis", "flavonoid biosynthesis" and "starch and sucrose metabolism". Our study also analyzed the expression patterns of SGs synthesis-related genes under two N regimes and the potential transcription factors linking N nutrition and SG metabolism. N-deficiency may promote SGs synthesis by changing the carbon metabolism flux or inducing certain transcription factors. Our results provide deeper insight into the relationship between N nutrition and SGs synthesis in stevia plants.