SummaryStaphylococcal enterotoxin (SE) ‐induced toxic shock is triggered by inflammatory cytokine signal amplification after SE binding to major histocompatibility complex class II molecules on antigen‐presenting cells and T‐cell receptors. Identifying host cellular elements contributing to this pro‐inflammatory signal amplification is critical for developing a strategy for therapeutic intervention. Myeloid differentiation primary‐response protein 88 (MyD88) is an intracellular signalling adaptor protein primarily known for mediating pro‐inflammatory cytokine responses. We investigated the role of MyD88 in staphylococcal enterotoxin A (SEA) ‐treated cell cultures and mouse models of toxic shock. Our results demonstrated that elevated levels of tumour necrosis factor‐α, interferon‐γ, interleukin‐1α/β (IL‐1α/β), IL‐2 and IL‐6 production correlated with up‐regulation of MyD88 after treatment of spleen cells and mice with SEA alone or in combination with lipopolysaccharide (LPS). The SEA‐induced lethality was also observed in (LPS‐independent) d‐galactosamine‐sensitized mice. While LPS potentiated SEA‐induced cytokine responses, d‐galactosamine treatment had no additive effect. Most importantly, our results demonstrated that MyD88−/− mice were resistant to SEA‐induced toxic shock and had reduced pro‐inflammatory cytokine responses. These results suggest that SEA‐induced lethality is primarily dependent on MyD88. Our findings offer an important insight on potential therapeutic treatment of SEA‐induced toxic shock targeting MyD88.