Objectives. Toll-like receptor (TLR)-9 recognizes unmethylated cytidine-phosphate:guanosine (CpG) motifs in bacteria. Therefore, the expression of TLR-9 may differ according to the results of bacterial culture, and thus a change in proinflammatory cytokine induction can also be expected. The authors aimed to assess the differences and relationships between the expression of TLR-9, cytokines, and nitric oxide synthase (NOS) in otitis media with effusion (OME) based on bacterial culture results. Methods. Sixty-eight patients with 01VPE were divided into culture-positive and culture-negative groups based on middle ear culture results. mRNA expression of TLR-9, NOS, and cytokines was measured and analyzed. Results. Bacteria were detected in 38.2% of patients, and the distribution was as follows: coagulase negative Staphylococcus (10.3%), Staphylococcus aureus (8.8%), Streptococcus pneumonia (5.9%), and Bacillus spp. and Haemophilus influenza combined (2.9%).There were no significant differences in epidemiologic characteristics according to the culture results. Down-regulation of TLR-9 was observed in the culture-positive group (P=0.019). Cytokines including interleukin (M)-12 (7=-0.582), tumor necrosis factor (TNF)-alpha (r=-0.569), interferon (IFN)-gamma (r=-0.442), IL-(7=-0.395) and inducible NOS (r=-0.256) tended to decrease with the detection of bacteria. Conclusion. The expression of TLR-9 significantly decreased in OME with confirmed bacterial pathogens. IL-12, TNF-alpha, IFN-f3, IL-6 expression tended to decrease with the detection of bacteria. The presence of bacterial pathogens in OME may be related to abnormalities in the innate immune system.