Anthrax lethal toxin triggers death in some cell types, such as macrophages, and causes a variety of cellular dysfunctions in others. Collectively, these effects dampen the innate and adaptive immune systems to allow Bacillus anthracis to survive and proliferate in the mammalian host. The diverse effects caused by the toxin have in part been attributed to its interference with signaling pathways in target cells. Lethal factor (LF) is the proteolytic component of the toxin, and cleaves six members of the mitogen-activated protein kinase kinase family after being delivered to the cytosol by the cell-binding component of the toxin, protective antigen. The effect of cleaving these mitogen-activated protein kinase kinases is to interfere with extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase signaling. Here, we characterized an LF mutant, LF-K518E/E682G, that was defective at causing pyroptosis in RAW 264.7 cells and at activating the Nlrp1b inflammasome in a heterologous expression system. LF-K518E/E682G did not exhibit an overall impairment of function, however, because it was able to downregulate the ERK pathway, but not the p38 or c-Jun N-terminal kinase pathways. Furthermore, LF-K518E/E682G efficiently killed melanoma cells, which were shown previously to undergo apoptosis in response to lethal toxin or to pharmacological inhibition of the ERK pathway. Our results suggest that LF-K518E/E682G is defective at cleaving a substrate involved in the activation of the Nlrp1b inflammasome.