Clostridium sordellii lethal toxin (TcsL) inactivates small GTPases via glucosylation and induces apoptosis in mammalian cells; however, signaling events that link substrate modification with modulation of the mitochondria in these cells has not been determined. Experiments in the current study examined TcsL modulation of the Akt signaling pathway and related downstream targets. Early in TcsL intoxication, cells demonstrated a dramatic decrease in phosphorylated Akt, and this event required toxin enzymatic activity. The decrease in phosphorylated Akt was followed by caspase-dependent processing of Bcl-x(L) and Bid, revealing the connection between GTPase inactivation and mitochondrial-mediated apoptosis observed in TcsL-intoxicated cells. Levels of glycogen synthase kinase-3beta declined during later times of TcsL intoxication, suggesting a second intermediate step in apoptosis. Collectively, these data provide insight into the cascade of signaling events that lead to apoptotic death of TcsL-intoxicated cells.