Under nutrient-deprived conditions, bacteria can grow in a tightly packed community and encase themselves in a protective polysaccharide matrix, a process called biofilm formation. Such a state provides the bacteria with protection against physical stresses and allows slowed growth and metabolism, which extends bacterial survival. In open environment such as water, EHEC respond to the phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoBR (1). The Pst (phosphate specific transport) system is involved in the Pi regulatory cascade and serves as a sensor of the extracellular Pi concentration. When the pstCAB genetic system is deleted, the regulator PhoB is constitutively activated and expression of genes from the Pho regulon is modulated (2,3). In this study we show that in EHEC pstCAB mutant, the capacity of biofilm formation and auto-agglutination was increased while motility was decreased (4). In double deletion mutant pstCAB and phoB, biofilm formation capacity, auto-agglutination and motility phenotypes were similar to wild type strain suggesting that PhoB is implicated. To identify Pho regulon members involved in biofilm formation we generated a Tn10 transposition mutant library derived from mutant pstCAB using pLOF/Cm that were screened for auto-agglutination and biofilm phenotypes. Of 5118 clones a total of 30 biofilm decreased mutants were selected. The transposon insertion site of mutants were identified by high throughput sequencing. Interestingly, several genes were involved in lipopolysaccharides (LPS) synthesis. Moreover our transcriptomic studies revealed that expression of glucuronic acid glycosyltransferase, waaH, responsible of LPS inner core modifications, was highly upregulated in Pi starvation condition and dependent of PhoB activation. This study highlighted the importance of extracellular (Pi) condition and Pho regulon in biofilm formation of EHEC. This may play a role in its transmission, persistence and virulence.