Background Staphylococcus aureus enterotoxins are known to induce an inflammatory response of the airways, increase sensitization to inhaled allergens, and decrease T-cell sensitivity to steroids. However, the effects of Staphylococcus enterotoxin A (SEA) on mucin secretion of airway epithelial cells still have not been reported. Therefore, in this study, the effect and brief signaling pathway of SEA on MUC5B expression were investigated in human airway epithelial cells. Methods In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of SEA on MUC5B expression were investigated using reverse transcriptase–polymerase chain reaction (PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). Results SEA increased MUC5B mRNA and protein expression. Toll-like receptor 2 (TLR2) mRNA expression was significantly increased after treatment with SEA. Knockdown of TLR2 by siRNA significantly blocked SEA-induced MUC5B mRNA expression. SEA significantly activated phosphorylation of extracellular signal–regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) significantly inhibited SEA-induced MUC5B mRNA expression. In addition, knockdown of ERK1/2 and p38 MAPK by siRNA significantly blocked SEA-induced MUC5B mRNA expression. Furthermore, the phosphorylation of ERK1/2 and p38 MAPK was significantly blocked by knockdown of TLR2 by siRNA. Conclusion These results show for the first time that SEA induces MUC5B expression via TLR2, ERK1/2, and p38 MAPK signaling pathway in human airway epithelial cells.