Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.