Low nitrogen stress impedes sustainable sorghum production by inhibiting plant growth and development. Our study explored the mitigating effects of exogenous brassinolide (BR) on sorghum seedlings under low N stress, focusing on growth, photosynthetic efficiency, chlorophyll fluorescence, and nitrogen metabolism. We found that low N significantly decreased seedling height, fresh and dry weights, and pigment content by 31.20% to 48.28%. BR application increased seedling height and biomass by 5.09% to 15.04% and 34.72% to 57.60%, respectively, compared to the low N condition. Photosynthetic parameters (Pn, Gs, Tr) declined under low N, with BR partially restoring these levels and reducing intercellular CO2 concentration (Ci). Chlorophyll fluorescence showed an increase in initial fluorescence (F0), maximum fluorescence (FM) and non-photochemical quenching (NPQ), but a decrease in maximum photochemical efficiency (Fv/Fm) under low N, which improved with BR supplementation. Ultrastructural analysis revealed BR preserved chloroplast integrity, reducing damage from N deficiency. BR also enhanced linear electron transport in PSI, and improved photochemical and non-photochemical quenching in PSI and PSII. Spectral reflectance indices confirmed improved light utilization efficiency in BR-treated seedlings. BR significantly increased key enzyme activities in N assimilation and elevated soluble protein, total N, ammonium, and nitrate contents. In summary, BR mitigate the adverse effects of nitrogen deficiency in sorghum by enhancing photosynthesis, nitrogen absorption, and preserving chloroplast structure and function. This research provides a significant theoretical foundation for optimizing fertilization strategies and holds substantial practical importance for the efficient utilization of infertile land and the achievement of high, stable yields in crop cultivation. (c) 2025 SAAB. Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.