BACKGROUND: The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is a critical molecule mediating interleukin-1β (IL-1β) responses. However, the role of the NLRP3 inflammasome in otitis media has not been fully examined. AIMS/OBJECTIVES: The purpose of this study is to assess the expression of NLRP3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain and a pyrin domain), and caspase-1 in lipopolysaccharide-induced otitis media. MATERIALS AND METHODS: BALB/c mice received a transtympanic injection of either lipopolysaccharide or phosphate-buffered saline. The mice were sacrificed 24 h after injection. Concentrations of IL-1β, NLRP3, ASC, and caspase-1 in the middle ear effusions were measured by enzyme-linked immunosorbent assay. Temporal bones were processed for histologic examination and immunohistochemistry. RESULTS: The transtympanic injection of lipopolysaccharide significantly upregulated levels of IL-1β, NLRP3, ASC, and caspase-1 in the middle ear as compared with the control mice. The proteins of NLRP3, ASC, and caspase-1 were observed in infiltrating inflammatory cells induced by lipopolysaccharide in the middle ear cavity. CONCLUSIONS AND SIGNIFICANCE: Lipopolysaccharide induces NLRP3 inflammasome components in the middle ear. The NLRP3 inflammasome may play an important role in the pathogenesis of otitis media. Modulation of inflammasome-mediated inflammation may be a novel therapeutic strategy for otitis media.