Otitis media (OM) in children is clinically important because of its detrimental effects on the development of language and motor coordination and is the most common reason for prescription of antibiotics. A recent bacteriological change in OM pathogens such as emergence of antibiotic resistance and vaccination-mediated pathogenic shift urges us to develop a new non-antibiotic strategy. The middle ear epithelium abundantly secretes a variety of antimicrobial molecules suppressing the viability of the common OM pathogens. Recently, we have demonstrated that the adenoviral vector is able to deliver the β-defensin 2 gene to the middle ear epithelial cells in vitro and in vivo, and adenovirus-mediated overexpression of β-defensin 2 is protective for experimental OM. There are many hurdles limiting successful clinical application of gene delivery to the respiratory epithelium of the tubotympanum; however, intratympanic gene therapy with β-defensin 2 is a promising alternative or adjuvant strategy for the management of OM.