Clostridium perfringens type C isolates cause necrotizing enteritis in humans and domestic animals. In vitro, type C isolates often produce beta toxin (CPB), beta2 toxin (CPB2), alpha toxin (CPA), perfringolysin O (PFO) and TpeL during (or after) late log-phase growth. In contrast, the current study found that many type C isolates respond to close contact with enterocyte-like Caco-2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco-2 cell-induced upregulation of CPB and PFO production involves the VirS/VirR two-component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression. However, the luxS quorum-sensing system is not required for the rapid upregulation of type C toxin production induced by contact with Caco-2 cells. These results provide the first indication of host cell:pathogen cross-talk affecting toxin production kinetics by any pathogenic Clostridium spp., identify in vivo versus in vitro differences in C. perfringens toxin expression, and implicate VirS/VirR as a possible contributor to some C. perfringens enteric diseases.