Abstract <p>Gram-negative bacteria cell-to-cell signaling, quorum sensing, has robust, well-understood molecular biological mechanisms: the <i>luxI</i>-type gene for signal synthesis and the <i>luxR</i>-type gene for signal reception. The evolution of this system remains veiled, while the signaling system has high specificity. To resolve the evolution of the <i>luxI</i>/<i>R</i>-system, three proteobacterial classes were selected, and their phylogeny based on the sequence was examined. Based on the ligand-docking simulation of <i>luxR</i>-type proteins, phylogeny focusing on functionality was also analyzed. Neutrality tests of sequence evolution were conducted on <i>luxI</i>- and <i>luxR</i>-type genes, using Tajima’s <i>D</i> and d<i>N</i>/d<i>S</i> ratio. Based on these analyses, the evolution of proteobacterial quorum sensing is proposed in which the <i>luxI</i>-type gene has undergone positive selection, showing different phylogeny compared to their pre-determined relationship. The <i>luxR</i>-type gene has undergone negative selection, which also regards that the conservation of most amino acids is critical to the functional reception, showing the relatively consistent phylogeny to pre-determined relationship. Compared to the sequence-based phylogeny, ligand-docking-based phylogeny showed the dependency on the signal usage of proteins, suggesting that negative selection of the <i>luxR</i>-type gene can still allow enough specificity and diversity of quorum sensing.</p>