AbstractInflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogenClostridium perfringensare nonredundant activators of the NLRP3 inflammasome in mice and humans.C. perfringenslecithinase (also known as phospolipase C) andC. perfringensperfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome‐dependent cytokines IL‐1β and IL‐18, and the induction of cell death independently of the pore‐forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin‐1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasomein vivoand that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase‐induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation duringC. perfringensinfection and that this mode of action can be similarly exploited for sensing by a single inflammasome.