Objective To explore the effect of IL-27 on pneumococcal clearance by regulating PI3K signaling pathway in acute otitis media (AOM).Methods A mouse model of AOM was established with Streptococcus pneumoniae infection. The mice were randomly divided into normal group, model group, experimental group and control group. Twelve hours before the infection with Streptococcus pneumoniae, the mice in the experimental group received retroauricular injection with 200 μL of IL-27(5 μg/mL), those in the control group with the same amount of PI3K specific inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-on (LY294002) (5 μg/mL), and those in the normal group and the model group with the same amount of normal saline. The mice in the model group, the experimental group and the control group were each injected with 5 μL of Streptococcus pneumoniae in the front and bottom of the eardrum, and the normal group was given the same dose of normal saline. Five days after the infection injection, an auditory brainstem response (ABR) test was performed. The number of inflammatory cells and bacteria in middle ear lavage fluids (MELF) was detected. ELISA was used to determine the expression of TNF-α and IL-6 in MELF. Hematoxylin-eosin (HE) staining was used to detect the pathological changes in the middle ear tissue. Immunohistochemistry and Western blot were used to detect the expressions of phosphorylation of phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated serine/threonine kinase (p-AKT) in the middle ear tissue.Results Compared with the normal group, the ABR threshold, the numbers of inflammatory cells and bacteria, the expressions of TNF-α, IL-6, p-PI3K, and p-AKT in the model group mice were significantly increased, and the AOM lesion pathology in the middle ear tissue was significantly increased. Compared with the model group, the ABR threshold, the numbers of inflammatory cells and bacteria, the expressions of TNF-α, IL-6, p-PI3K, and p-AKT in the experimental and control mice were significantly reduced, and the pathology of AOM damage in the middle ear tissue was significantly reduced. The differences were all statistically significant (all P<0.05).Conclusion In AOM with Streptococcus pneumoniae infection, IL-27 can regulate the neutrophil clearance of pathogens by inhibiting the phosphorylation of PI3K signal.