Abstract Intracellular sensing of LPS is crucial for antimicrobial defense and sepsis. Noncanonical inflammasome sensing of cytosolic LPS via caspase-4/11 triggers gasdermin-D mediated pyroptosis and the release of cytokines and alarmins. During infection, LPS associated with bacteria or bacterial outer membrane vesicles gains access to the cytosol. However, free LPS can access the cytosol in vivo. The mechanism by which free LPS gains access to the cytosol and activate the caspase-11-noncanonical inflammasome in vivo mostly remains elusive. CD14, a GPI-anchored glycoprotein, binds and transfers LPS to the TLR4-MD2 complex. While the role of CD14 in TLR4-mediated host responses to extracellular LPS is clear, its role in the noncanonical inflammasome sensing of cytosolic LPS is poorly defined. Here, we found that cytosolic LPS-induced GSDMD cleavage, IL-1β, IL-18 and DAMP release, pyroptosis and lethality were remarkably impaired in CD14-deficient mice, but not in TLR4-deficient mice. By using Cd14−/− and Casp11−/− mice strains on a Tlr4−/− background, we have decoupled CD14’s role in caspase-11 activation from its known function in TLR4 signaling. Mechanistically, CD14 mediates caspase-11 activation by facilitating the internalization of LPS in a TLR4-independent manner. Overall, our findings highlight a new TLR4-independent role of CD14 in noncanonical inflammasome activation by intracellular LPS in vivo.