In the present study, by using NTHi-induced otitis media and pneumonia model in wild type (WT) and Cyld-deficient mice, we provide in vivo evidence that NTHi induces pro-inflammatory response through TLR2-dependent MyD88-TRAF6/7-NF-κB signaling pathway, and CYLD negatively regulates NF-κB-dependent inflammatory response by NTHi via deubiquitination of TRAF6 and TRAF7. Here, TLR2 is linked to otitis media.