<p>Umami peptides are frequently reported in fermented foods, yet the sequence organization principle that determines their functional umami intensity remains unclear. Using shrimp paste as a model system, we demonstrate that umami-enhancing activity of peptides is determined not merely by amino acid composition, but critically by their sequential organization. Specifically, umami-active peptides consistently form an acidic-polar core at positions N2-N4, especially by Asp and Glu residues that provide sustained negative charge and high polarity. In contrast, bulky hydrophobic residues and strongly cationic residues are systematically excluded from this central region and preferentially shifted toward the C-terminus, preventing disruption of the core organization. Based on this principle, nine octapeptides were rationally designed with graded conformity to the proposed organization principle. Across receptor-binding fluorescence assays, sensory evaluation, and molecular docking and dynamics simulations, peptides that more closely followed this sequence organization principle consistently exhibited enhanced receptor interaction stability and stronger umami intensity. External validation using reported umami peptides further confirmed that the proposed principle can explain sequence–function relationships beyond the designed peptide set. These results establish a clear, reproducible sequence organization principle underlying umami peptide activity and advance the field from descriptive peptide identification toward principle-guided sequence design.</p>