Group A streptococci (GAS) produce several secreted products that are thought to enhance pathogenicity by facilitating spread of the organisms through host tissues. Two such products, streptolysin O (SLO) and NAD+-glycohydrolase, appear to be functionally linked, in that SLO is required for transfer of NAD+-glycohydrolase into epithelial cells. However, the effects of NAD+-glycohydrolase on host cells are largely unexplored. We now report that SLO-mediated delivery of NAD+-glycohydrolase to the cytoplasm of human keratinocytes results in major changes in host cell biology that enhance GAS pathogenicity. We derived isogenic mutant strains deficient in the expression of SLO, NAD+-glycohydrolase or both proteins in the background of a virulent, M-type 3 strain of GAS. All three mutant strains were internalized by human keratinocytes more rapidly and in higher numbers than were organisms from the wild-type strain. Association of the mutant strains with keratinocytes also resulted in reduced cytotoxicity and reduced keratinocyte apoptosis compared with wild-type GAS. These results support a model in which NAD+-glycohydrolase contributes to GAS pathogenesis by modulating host cell signalling pathways to inhibit GAS internalization, to augment SLO-mediated cytotoxicity and to induce keratinocyte apoptosis. We conclude that NAD+-glycohydrolase is a novel type of bacterial toxin that acts intracellularly in the infected host to enhance the survival and proliferation of an extracellular pathogen.