Repair of plasma membranes damaged by bacterial pore-forming toxins, such as streptolysin O or perfringolysin O, during septic cardiomyopathy or necrotizing soft tissue infections is mediated by several protein families. However, the activation of these proteins downstream of ion influx is poorly understood. Here, we demonstrate that following membrane perforation by bacterial cholesterol-dependent cytolysins, calcium influx activates mixed lineage kinase 3 independently of protein kinase C or ceramide generation. Mixed lineage kinase 3 uncouples mitogen-activated kinase kinase (MEK) and extracellular-regulated kinase (ERK) signaling. MEK signals via an ERK-independent pathway to promote rapid annexin A2 membrane recruitment and enhance microvesicle shedding. This pathway accounted for 70% of all calcium ion-dependent repair responses to streptolysin O and perfringolysin O, but only 50% of repair to intermedilysin. We conclude that mixed lineage kinase signaling via MEK coordinates microvesicle shedding, which is critical for cellular survival against cholesterol-dependent cytolysins.