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Inorganic polyphosphate (polyP) is a universally conserved polymer involved in various biological processes, but its role as a direct protein regulator remains largely unexplored. Lysine polyphosphate modification (KPM), a strong but non-covalent interaction between polyP and lysine-rich protein sequences, has not been functionally characterized until now. In this study, we present the first investigation into KPM's biological significance using <i>Pseudomonas aeruginosa</i>, a critical priority pathogen known for its antibiotic resistance and virulence. We identified two essential bacterial proteins, EngA and SrmB, as novel KPM targets. Through site-specific lysine deletions, we demonstrated that disrupting lysine-polyP interactions severely impairs biofilm formation and significantly reduces the production of key virulence factors, including pyoverdine and pyocyanin. These findings establish a direct functional link between polyP and bacterial pathogenicity mediated by KPM. Our results highlight KPM as a previously unrecognized regulatory mechanism critical for controlling bacterial virulence factors. This work uncovers the first functional role of KPM and its importance in regulating virulence phenotypes in a major human pathogen.IMPORTANCEPolyphosphate is commonly known for its roles in metabolism and stress response. How inorganic polyphosphate (polyP) facilitates bacterial virulence has remained largely elusive. This study reveals that lysine polyphosphate modification (KPM), a chemical interaction between polyP and lysine-rich proteins, is essential for bacterial survival and pathogenicity in <i>P. aeruginosa</i>, a harmful microbe responsible for difficult-to-treat infections. We discovered that disrupting KPM in key proteins impairs the bacteria's ability to form protective biofilms and produce harmful toxins. This previously unknown biological process links polyP to protein function in controlling bacterial virulence factors. Our findings open new possibilities for developing anti-virulence therapies aimed at reducing bacterial infections without promoting antibiotic resistance.