Clostridium perfringens is the causative agent of human clostridial myonecrosis and food poisoning. It is able to produce more than 20 different toxins, of which a-toxin and perfringolysin O have been shown to be involved in myonecrotic disease. Our previous work has shown that the RevSR two-component regulatory system is involved in regulating virulence in our mouse myonecrosis model. Microarray and RNAseq analysis of a revR mutant showed that expression of the genes encoding a-toxin and perfringolysin O were not significantly altered, implying that other factors may also play a role in virulence. The RNAseq data showed that the expression of the engCP gene (previously CPE0693) was significantly down-regulated in a revR mutant. A previous study had shown that EngCP had endo a-N-acetylgalactosaminidase activity and therefore could hydrolyse O-glycans. Similar enzymes from several Gram-positive pathogens have been postulated to contribute to their virulence. Consequently, we constructed an engCP Targetron mutant of C. perfringens and tested this mutant in the mouse myonecrosis model. The results showed that the engCP mutant was less virulent compared to its wild-type parent strain and that virulence could be restored by complementation in trans with the wild-type engCP gene. To examine its function, we demonstrated that purified EngCP could deglycosylate a-dystroglycan, a heavily O-glycosylated protein located in the sarcolemmal membrane of myofibres. However, EngCP did not appear to enhance membrane damage in C. perfringens-infected muscle tissue. Glycan array analysis of EngCP indicated that it could bind to glycan structures found on blood group antigens that are associated with immune cells, suggesting that EngCP may play a role in immunomodulation. These data provide evidence that EngCP is required for virulence in C. perfringens and indicate that although the toxins are important for disease, other factors also play a significant role in the disease process.