The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis.