Background: Otitis media is the most common disease of childhood, and represents an important health challenge to the 10-15% of children who experience chronic/recurrent middle ear infections. The middle ear undergoes extensive modifications during otitis media, potentially involving changes in the expression of many genes. Expression profiling offers an opportunity to discover novel genes and pathways involved in this common childhood disease. Results: Approximately 8% of the sampled transcripts defined the signature of acute NTHi-induced otitis media across time. Hierarchical clustering of signal intensities revealed several temporal gene clusters. Network and pathway enrichment analysis of these clusters identified sets of genes involved in activation of the innate immune response, negative regulation of immune response, changes in epithelial and stromal cell markers, and the recruitment/function of neutrophils and macrophages. We also identified key transcriptional regulators related to events in otitis media, which likely determine the expression of these gene clusters. A list of otitis media susceptibility genes, derived from genome-wide association and candidate gene studies, was significantly enriched during the early induction phase and the middle re-modeling phase of otitis but not in the resolution phase. Our results further indicate that positive versus negative regulation of inflammatory processes occur with highly similar kinetics during otitis media, underscoring the importance of anti-inflammatory responses in controlling pathogenesis. Conclusions: The results characterize the global gene response during otitis media and identify key signaling and transcription factor networks that control the defense of the middle ear against infection. These networks deserve further attention, as dysregulated immune defense and inflammatory responses may contribute to recurrent or chronic otitis in children.