<p>Plants depend on nitrogen (N) for their growth, development, and metabolic functions. However, the regulatory mechanisms modulating N assimilate allocation under varying N forms are unclear. This study examines N metabolism and spatial distribution in maize seedlings subjected to four N treatments (T1 to T4): T1, 1 mM NO₃⁻ (sole NO₃⁻); T2, substitution of 1 mM NO₃⁻ with 1 mM NH₄⁺ (N form substitution, NFS); T3, 1 mM NH₄⁺ (sole NH₄⁺); and T4, 0.5 mM NH₄NO₃ (mixed N supply). The NFS treatment induced significant physiological and molecular adaptations, such as enhanced growth and total biomass under fluctuating N conditions. NFS-treated plants exhibited improved photosynthesis, increased protein and amino acid synthesis, and increased NO₃⁻ and NH₄⁺ accumulation. Activities of key N metabolism enzymes, such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT), were significantly upregulated, supporting efficient assimilation of both NO₃⁻ and NH₄⁺. Furthermore, spatial and diurnal analyses revealed dynamic N partitioning and adaptive regulation, with NFS-treated plants maintaining consistently higher NO₃⁻ and NH₄⁺ levels in leaves, roots, sheaths, and developing ears. These findings highlight the robust plasticity of maize N metabolism under NFS conditions and provide valuable insights into optimizing N use efficiency (NUE) for sustainable crop production. Future studies will focus on exploring these adaptive mechanisms across different maize genotypes and under field conditions to improve NUE and productivity in varying N environments.</p>