Abstract Neisseria meningitidis causes acute severe diseases, including sepsis and meningitis, and more benign manifestations such as chronic meningococcemia or colonization of the upper respiratory tract. The inflammatory response, which contributes to the pathogenesis of meningococcal disease, is initiated by pattern recognition receptors, among which Toll‐like receptors (TLR)s have been ascribed a particularly important role. We have previously demonstrated that N. meningitidis induce proinflammatory cytokine expression through TLR2 and TLR4. Here we characterize the molecular basis for differential activation of the inflammatory response by two N. meningitidis strains. This difference was due to differential ability to activate signal transduction through TLR4, as HEK293 cells expressing TLR4 produced significantly different levels of interleukin‐8 in response to these strains. At the level of signal transduction, the two strains differed substantially in their ability to activate the pathway to nuclear factor κB in HEK293‐TLR4/MD2 cells at late, but not early, time points. TLR4 activates two signal transduction pathways: one dependent on the adaptor molecule MyD88 and one independent of MyD88, and these pathways induce distinct patterns of gene expression in response to TLR4 ligands. By using macrophages from TLR2−/− mice, we observed that the two strains differed in their ability to activate the TLR4‐induced MyD88‐independent pathway, but not the MyD88‐dependent pathway. This idea was further supported by experiments where either of the two pathways was inhibited and IL‐8 secretion was measured. These data therefore provide molecular insight into activation of the inflammatory response by N. meningitidis, which is one of the key events in the pathogenesis of meningococcal disease.