We analysed the region encoding VR‐RNA (VirR‐regulated RNA), which has been reported to be positively regulated by the two‐component VirR/VirS system in Clostridium perfringens. The VR‐RNA promoter identified by primer extension analysis was preceded by a probable VirR‐binding site (CCAGTTNNNCAC), which resembled a repeated sequence motif present in the promoter region of the theta‐toxin (pfoA) gene. A VR‐RNA‐null mutant, constructed by a homologous recombination, exhibited a reduced amount of transcription of the alpha‐ (plc) and kappa‐toxin (colA) genes, which was restored by the complementation of intact VR‐RNA, indicating that the VR‐RNA region plays an important role in the regulation of the plc and colA genes in C. perfringens. It was found that the regulatory effect was observed even when the hyp7 gene encoded on VR‐RNA was deleted or a nonsense mutation was introduced in the hyp7‐coding region. We found that the small 3′‐portion of VR‐RNA was sufficient for the activation of toxin genes, which suggested that VR‐RNA itself could act as an RNA regulatory molecule for the plc and colA genes mediating the regulatory information from the VirR/VirS system in C. perfringens.