Soybean production contributes to ca. 60% of global plant-based protein used for food and feed. Brazil is the largest soybean producer and exporter, with 60% from tropical and 40% from subtropical environments. Nitrogen (N) can play an essential role in the storage of proteins in seeds; thus, it could be a key factor in increasing the quantity and quality of seeds in high-yielding soybean crops. Unlike in temperate environments, there is a gap of knowledge on whether soybean grown under tropical and subtropical climates are limited by N-fertilization to sustain the seed yield increase without detriments in seed protein concentration. This study aimed to evaluate the effect of N-fertilization on soybean seed yield, protein and oil concentrations in tropical and subtropical environments in Brazil, thus contributing to agricultural intensification procedures and food security studies. Two levels of N-fertilization (0 and 1000 ka/ha) were tested across 11 tropical or subtropical environments. The range of latitudes explored here was from 12°S to 29°S, representing the major soybean-producing regions in Brazil either under rainfed or irrigated conditions. We found that seed yield responses to N-fertilization were significant (in some environments under rainfed with an average increase of 7%) or not significant (in irrigated). Seed protein increases due to improved N-fertilization (on average 4% for irrigated and 12% for rainfed conditions) were much higher than previous reports from temperate environments. Regardless of N supply and water deficit, there was a trend of seed protein and oil concentration increasing toward lower latitudes.