Nitrogen (N) based secondary metabolite production is thought to be costly to plants because N is required for growth, as well as, the synthesis of these compounds. Therefore, variation in N availability may result in variation in N-based secondary metabolite production. Here, we determine the effect of N fertilization on caffeine (N-based alkaloid) production in coffee (Coffea arabica) seedlings. A growth chamber experiment was performed with three N treatments applied to seedlings. N fertilization increased plant growth, leaf biomass, and plant N. Caffeine concentration in phloem exudates was greater in high-N fertilized plants relative to intermediate-and low-N plants. However, leaf, stem, root, and total overall caffeine concentration and content did not differ across N treatments. These results suggest caffeine in coffee is strongly regulated by genetic factors, and environment is likely less important to caffeine phenotype. This is among the first studies to investigate the effect of N fertilization on caffeine within the phloem, which has important implications for herbivores that are sensitive to caffeine and plant N and feed from the phloem of coffee.