There is now ample evidence that bacterial virulence is modulated by eukaryotic messengers including hormones. We have shown previously that the C-type Natriuretic Peptide (CNP), a peptide produced by lung, enhances Pseudomonas virulence. In the present work, we studied the effect of CNP on bacterial biofilm formation and we looked for a putative P. aeruginosa protein that could explain the effect of CNP on bacteria. We used in silico and in vitro approaches to identify a bacterial sensor for CNP. Additionally, pharmacological tools were used as an innovative strategy to characterize the binding site in P. aeruginosa. We observed that CNP inhibits P. aeruginosa biofilm formation. This effect is totally prevented by Isatin, an antagonist of natriuretic peptide receptors in eukaryotic cells. The actions of CNP on P. aeruginosa virulence are mimicked by the eukaryotic receptor NPR-C agonist cANF4-23. Screening and comparing 3D structures of human natriuretic peptides receptors and Pseudomonas proteins revealed that the bacterial protein AmiC shows significant homology with the human C-type eukaryotic natriuretic peptide receptor (hNPR-C) and that both CNP and Isatin can interact with AmiC using the same amino acids as in hNPR-C. Finally, recombinant protein AmiC was purified and the protein interactions assessed using MicroScale Thermophoresis. The results showed that both CNP and hNPR-C agonist bind AmiC protein with a KD of 2 µM and less than 100 nM respectively, whereas hNPR-A agonist has poor affinity for AmiC. Using an amiC- mutant strain and its complemented derivative, we definitively validated the crucial role of AmiC protein on CNP’s effect on P. aeruginosa biofilm formation. As observed for hNPR-C in mammals, AmiC is highly selective and can discriminate between the different natriuretic peptides, since another natriuretic peptide (brain natriuretic peptide, BNP) has no affinity with AmiC. In conclusion, our work provides the first demonstration that the bacterial protein AmiC may be an ortholog of the eukaryotic receptor hNPR-C, acting as a CNP sensor in P. aeruginosa. AmiC appears to modulate a switch between chronic and acute infection phenotypes depending on exposition to host factors. The observation that CNP strongly decreases bacterial biofilm formation should have major consequences for cystic fibrosis treatment.