Several quorum sensing systems occurring in Bacillus subtilis, e.g. Rap-Phr systems, were reported to interact with major regulatory proteins, such as ComA, DegU, and Spo0A, in order to regulate competence, sporulation, and synthesis of secondary metabolites. In this study, we characterized a novel Rap-Phr system, RapA4-PhrA4, in Bacillus velezensis NAU-B3. We found that the rapA4 and phrA4 genes were co-transcribed in NAU-B3. When rapA4 was expressed in the heterologous host Bacillus subtilis OKB105, surfactin production and sporulation were severely inhibited. However, when the phrA4 was co-expressed, the RapA4 activity was inhibited. The transcription of the surfactin synthetase srfA gene and sporulation-related genes were also regulated by the RapA4-PhrA4 system. In vitro results obtained from electrophoretic mobility shift assay (EMSA) proved that RapA4 inhibits ComA binding to the promoter of the srfA operon, and the PhrA4 pentapeptide acts as anti-activator of RapA4. We also found that the F24 residue plays a key role in RapA4 function. This study indicated that the novel RapA4-PhrA4 system regulates the surfactin synthesis and sporulation via interaction with ComA, thereby supporting the bacterium to compete and to survive in a hostile environment. KEY POINTS: •Bacillus velezensis NAU-B3 has a novel Rap-Phr quorum sensing system, which does not occur in model strains Bacillus subtilis 168 and B. velezensis FZB42. •RapA4-PhrA4 regulates surfactin production and sporulation. •RapA4-PhrA4 interacts with the ComA protein from ComP/ComA two-component system.