ABSTRACT Cell suspension culture has been used as a model to study metabolic changes to several stresses. To have detailed information of nitrogen (N) limitation on sugarcane metabolism, a controlled study of the primary metabolites and representative compounds of secondary metabolism was developed using suspension cells growing under three different N regimes: normal condition (40 mmol·L–1 NO-3), slightly deficient (12 mmol·L–1 NO-3) and completely deficient (0 mmol·L–1 NO-3). Sugarcane cells were harvested after 3 and 7 days of treatment. A range of changes in the levels of amino acids, organic acids, sugars and phenolic compounds were observed upon the growth conditions applied. Nitrogen limitation remarkably affected the amino acids and carbohydrates biosynthesis, which, associated with the changes observed on phenolic compounds contents, indicates the upregulation of carbon sink compensation mechanisms in these sugarcane cells exposed to N starvation. As expected, the results showed that N limitation might cause an extensive metabolic reprogramming of both carbon and N metabolism in sugarcane cells, and these changes are related to the intensity of the starvation. Nitrogen is essential for plant growth and development, and its limitation sharply reduces crop yield. Thus, these results open new perspectives for in planta studies concerning carbon and N metabolisms balance in this crop.