Clostridium difficile virulence requires secretion of two exotoxins: TcdA and TcdB. The precise mechanism of toxin uptake and delivery is undefined, but current models predict that the cysteine protease domain (CPD)-mediated autocleavage and release of glucosyltransferase domain (GTD) are crucial for intoxication. To determine the importance of CPD-mediated cleavage to TcdB cytotoxicity, we generated two mutant toxins – TcdB-C698S and TcdB-H653A – and assayed their abilities to intoxicate cells. The CPD mutants include an intact GTD but lack the cysteine protease activity. The mutants had reduced potency in that their effect on cells was delayed and required higher concentrations than wild-type TcdB. They did eventually cause cell rounding, glucosylation of Rho GTPases, and apoptosis that was indistinguishable from that caused by TcdB. Although the mutant toxins caused a complete cell rounding, they failed to release their GTD into cytosol, whereas wild-type TcdB displayed significant autocleavage and release of GTD. We conclude that the cysteine protease-mediated autocleavage and release of GTD is not a prerequisite for the cytotoxic activity of TcdB, but rather limits the potency and speed of Rho GTPase glucosylation. Our findings revise and refine the current model for the mode of the action and cellular trafficking of TcdB.