Mucoid otitis media (MOM) is characterized by viscous fluid, high in mucin concentration, which accumulates in the middle ear cavity. Recent studies suggest that initial infection in the middle ear cleft may be key to the development of MOM. However, factors of the initial infection attributed to the stimulation of mucin production are not clearly understood. This study demonstrated that tumor necrosis factor (TNF)-α, a proinflammatory cytokine in mucoid effusion, markedly increased Muc2 mucin mRNA expression in middle ear epithelium, in a time- and dose-dependent manner. Parallel to this was a marked increase in mucin glycoprotein in middle ear fluid. Also, TNF-α demonstrated an autocrine and/or paracrine effect on the expression of endogenous TNF-α gene in the middle ear, which may contribute to the production of mucin in this study. These findings suggest that TNF-α plays an important role in the development of MOM by stimulating mucin metabolism.