Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.