TcdB, an intracellular bacterial toxin that inactivates small GTPases, is a major Clostridium difficile virulence factor. Recent studies have found that TcdB produced by emerging/hypervirulent strains of C. difficile is more potent than TcdB from historical strains, and in the current work, studies were performed to investigate the underlying mechanisms for this change in TcdB toxicity. Using a series of biochemical analyses we found that TcdB from a hypervirulent strain (TcdBHV) was more efficient at autoprocessing than TcdB from a historical strain (TcdBHIST). TcdBHV and TcdBHIST were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdBHV. Using an activity-based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdBHIST was identified, while only a processed form of TcdBHV could be detected under the same conditions. Using a much higher concentration (200 µM) of the probe revealed an activated uncleaved form of TcdBHV, indicating a preferential and more efficient engagement of intramolecular substrate than TcdBHIST. Furthermore, a peptide-based inhibitor (Ac-GSL-AOMK) was found to block the cytotoxicity of TcdBHIST at a lower concentration than required to inhibit TcdBHV. These findings suggest that TcdBHV may cause increased cytotoxicity due to more efficient autoprocessing.