Nitrogen (N) is a major component of the photosynthetic apparatus and is widely used as a fertilizer in crops. However, to the best of our knowledge, the dynamic of photosynthesis establishment due to differential N supply in the bioenergy crop sugarcane has not been reported to date. To address this question, we evaluated physiological and metabolic alterations along the sugarcane leaf in two contrasting genotypes, responsive (R) and nonresponsive (NR), grown under high- and low-N conditions. We found that the N supply and the responsiveness of the genotype determined the degree of senescence, the carboxylation process mediated by phosphoenolpyruvate carboxylase (PEPcase) and differential accumulation of soluble sugars. The metabolite profiles indicated that the NR genotype had a higher respiration rate in the youngest tissues after exposure to high N. We observed elevated levels of metabolites related to photosynthesis in almost all leaf segments from the R genotype under high-N conditions, suggesting that N supply and the ability to respond to N influenced photosynthesis. Therefore, we observed that N influence on photosynthesis and other pathways is dependent on the genotype and the leaf region.