Otitis media (OM), a common infectious disease in children, is associated with bacterial middle ear (ME) infection. Toll-like receptors (TLRs) are important mediators of innate immune responses, and TLR9 specifically recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motifs in bacterial DNA. Additional sensors of foreign DNA have recently been identified. The role of DNA sensing and TLR9 was investigated in a murine model of OM induced by non-typeable Haemophilus influenzae (NTHi). Expression of genes related to DNA-sensing pathways involved in innate immunity was assessed via DNA microarray, qPCR and immunohistochemistry. Middle ear responses to NTHi were examined in wild-type and TLR9(-/-) mice by histopathology and bacterial culture. Expression of TLR9 signaling genes was modestly up-regulated during OM, as was TLR9 protein in both ME mucosal cells and infiltrating leukocytes. However, genes known to be regulated by CpG DNA were dramatically up-regulated, as were genes involved in DNA sensing by DIA, Pol-III and AIM2. Toll-like receptor 9 deletion significantly prolonged the inflammatory response induced by NTHi in the ME and delayed bacterial clearance. The results suggest that DNA sensing via TLR9 plays a role in OM pathogenesis and recovery. Alternative forms of DNA sensing may also contribute to OM.