Cyclic di-GMP (c-di-GMP) has been found to be widely present in bacterial kingdom, acting as a secondary messenger to control many important cellular activities, such as biofilm formation, biogenesis and action of flagella and pili, and synthesis and secretion of pathogenic factors in diverse bacteria. C-di-GMP is generated by the GGDEF domain-containing diguanlyate cyclases and degraded by the EAL domain- or HD-GYP domain-containing phosphodiesterases. The mechanisms of c-di-GMP biosynthesis and degradation have been characterized in some detail. However, the nature of c-di-GMP receptors and the mechanisms of c-di-GMP-mediated regulation are still not fully understood. In the past several years, a variety of c-di-GMP-binding protein receptors and two RNA-based riboswitches have been described but it is clear that many more remain to be identified. In the past, the PilZ domain is possibly the most important c-di-GMP binding domain, and has been found to be present in association with many different protein domains to carry out a variety of c-di-GMP regulated processes. However, we have found that although PilZ or other reported c-di-GMP binding domains have been intensively studied, there are many bacteria that completely lack any such c-di-GMP binding domains. It indicates that there are other c-di-GMP binders that are not discovered yet. Recently, we have found one novel c-di-GMP binding domain that uses anintriguing binding modecompletely different to anyc-di-GMP binders reported to date. It exhibits a 53-residue motif for recognizing and binding c-di-GMP, using many hydrophobic residues that are highly conserved. It also uses unique peptide backbone amide protons to form two hydrogen-bonds with the c-di-GMP guanine via its Hoogsteen-edge. In addition, it exhibits a stronger binding affinity than PilZ domains, approximately ten fold stronger. Importantly, such a strong binding stems mostly from hydrophobic interactions, not via polar interactions. Thus the discovery of this novel c-di-GMP binding mode will sparkle the next phase of c-di-GMP research.