Clostridium perfringens β-toxin (CPB) is a prototypic hemolysin β-Pore-Forming Toxin (βPFT) involved in the development of necrotic enteritis in animals and humans. This highly potent exotoxin targets cells via binding to its membrane receptor, the Ig6 domain of Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) and formation of an ion permissive transmembrane pore complex (Bruggisser et al., 2020). The previously determined cryo-EM structure of the CPB pore complex highlighted several flexible loops in the rim domain that are unique to CPB compared to other hemolysin βPFTs (Bruggisser, Iacovache et al., 2022). Based on this, we hypothesized that these loops constitute the receptor binding site of CPB and are responsible for the toxin’s remarkable receptor specificity. To test this, we generated a series of recombinant CPB mutants with single and multiple point mutations as well as a partial domain exchange with other hemolysin βPFTs. Our results show that two flexible loops in the rim domain of CPB are essential for receptor interaction and toxicity. These findings provide valuable insights for further research on related toxins and open new avenues for designing anti-virulence strategies against important clostridial exotoxins.