Clostridium perfringens is the causative agent of human clostridial myonecrosis and food poisoning. Previously, we identified an orphan response regulator, RevR, which when mutated resulted in virulence attenuation in the mouse myonecrosis model. RevR has sequence identity to the PhoB family of response regulators and is predicted to contain a helix-turn-helix DNA binding motif. The revR gene is situated downstream of genes encoding a putative phosphate uptake (pst) operon. RNAseq and previous microarray analysis showed that the pst genes were down-regulated in a revR mutant. Gel mobility shift experiments showed that RevR regulates the expression of these genes by binding to the promoter region, while fluorescence-based DNase I footprinting demonstrated that the DNA sequence recognised by RevR had organisational and sequence similarity to Pho boxes from other bacteria. Bioinformatic analysis suggested that the CPE1757 orphan sensor histidine kinase, which we have designated as RevS, was a homolog of the PhoR sensor histidine kinase. Interaction between RevR and RevS was predicted using two different algorithms and subsequently was confirmed experimentally with a bacterial two hybrid system. Interaction was also detected between RevS and PhoU, a regulator of the phosphate uptake system in other bacteria. Finally, we mutated the revS gene and showed that the resultant mutant was attenuated for virulence, to levels similar to that of the revR mutant. In conclusion, we have shown that RevR binds to Pho boxes, indicating that it is potentially part of a phosphate regulon, and we have identified its cognate sensor histidine kinase, RevS. We now have identified the two essential parts of a new regulatory network that is involved in the regulation of virulence in C. perfringens.