The inflammatory cytokine IL-1beta is critical for host responses against many human pathogens. Here, we define Group B streptococcus (GBS)-mediated activation of the Nod-like Receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, beta-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1beta production.