The innate immune response to Listeria monocytogenes depends on phagosomal bacterial degradation by macrophages. Here, we describe the role of LIMP-2, a lysosomal type III transmembrane glycoprotein and scavenger-like protein, in Listeria phagocytosis. LIMP-2-deficient mice display a macrophage-related defect in Listeria innate immunity. They produce less acute phase pro-inflammatory cytokines/chemokines, MCP-1, TNF-α, and IL-6 but normal levels of IL-12, IL-10, and IFN-γ and a 25-fold increase in susceptibility to Listeria infection. This macrophage defect results in a low listericidal potential, poor response to TNF-α activation signals, impaired phago-lysosome transformation into antigen-processing compartments, and uncontrolled LM cytosolic growth that fails to induce normal levels of acute phase pro-inflammatory cytokines. LIMP-2 transfection of CHO cells confirmed that LIMP-2 participates in the degradation of Listeria within phagosomes, controls the late endosomal/lysosomal fusion machinery, and is linked to the activation of Rab5a. Therefore, the role of LIMP-2 appears to be connected to the TNF-α-dependent and early activation of Listeria macrophages through internal signals linking the regulation of late trafficking events with the onset of the innate Listeria immune response.