Background and Aims: Nitrogen (N) distribution in plants is intricately linked to key physiological functions, including respiration, photosynthesis, structural development, and nitrogen storage. However, the specific effects of different N morphologies on N accumulation and plant growth are poorly understood. Our research specifically focused on determining how different N morphologies affect N absorption and biomass accumulation., Methods: This study elucidated the impact of different application rates (CK: 0 g N/plant; T1: 4 g N/plant; T2: 8 g N/plant) of N fertilizer on N and biomass accumulation in tobacco cultivars Hongda and K326 at different growth stages., Results: Our findings emphasize the critical role of N distribution in various plant parts, including leaves, stems, and roots, in determining the complex mechanisms of N and biomass accumulation in tobacco. We found that in relation to total N, a greater ratio of water-soluble N ( N w ) in leaves facilitated N accumulation in leaves. In contrast, an increased ratio of SDS (detergent)-insoluble N ( N in-SDS ) in leaves and non-protein N ( N np ) in roots hindered this increase. Additionally, our results indicate that a greater proportion of N np in leaves has a negative impact on biomass accumulation in leaves. Furthermore, elevated levels of N in-SDS , N w , and N np in roots, and N np in leaves adversely affected biomass accumulation in tobacco leaves. The Hongda cultivar exhibited greater biomass and N accumulation abilities as compared to K326., Conclusions: Our findings highlight the significant role of distribution of N morphologies on plant growth, as well as N and biomass accumulation in tobacco plants. Understanding N distribution allows farmers to optimize N application, minimizing environmental losses and maximizing yield for specific cultivars. These insights advance sustainable agriculture by promoting efficient resource use and reducing environmental impact., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Jiang, Ahmed, Yang, Yang, Zhang, Mei, Alharbi, Shan, Guo and Zhao.)