Staphylococcal 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-alpha, interferon-gamma, interleukin-1alpha/beta (IL-1alpha/beta), 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.