Otitis media with effusion (OME) often occurs in infants and young children, which is related to allergic reactions. Notch signaling pathway plays an important role in allergic responses. In this study, we aimed to investigate the role of Notch signaling pathway in the ovalbumin (OVA)-mediated allergic OME in vivo. OVA-induced OME rats were treated with a control vehicle or a gamma-secretase inhibitor, N4N-(3,5-difluorophenacetyl)-L-alanyfl-S-phenylglycine t-butyl ester (DAPT) suppressing the Notch signaling. We studied the effect of Notch signaling pathway in OME model, including histopathological assessment, the expression of Th1 cytokines (IFN-gamma), Th2 cytokines (IL-4, IL-5), key transcription factors (T-bet, GATA-3) by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the level of Notch ligand (Jagged1) and the downstream target gene Hes1 were also evaluated by qRT-PCR and immunofluorescent staining. We observed that the production of Th2 cytokines was increased, the level of Th1 cytokines was decreased in OME experimental model. Likewise, Th2-cytokine(IL-4)level was reduced, but the level of Th1 cytokines(IFN-gamma) was no changes. Additionally, administration of DAPT induced a decrease in the expression of GATA-3 mRNA, however, no influence on T-bet mRNA production. These results suggest that there is an imbalance with Th1/Th2 in OVA-mediated allergic OME. DAPT treatment can block the Notch signaling pathway and relieve the middle ear inflammation through modulating the level of Th2 responses in OVA-induced allergic OME.