This study was to explore a novel IL-33/ST2/IL-9/IL-9R signaling pathway that disrupts ocular surface barrier and amplifies allergic inflammation. Two murine models of experimental allergic conjunctivitis (EAC) and IL-9 topical challenge in wild type Balb/c and ST2-/- mice, and two culture models of primarily human corneal epithelial cells (HCECs) and mouse CD4+ T cells were performed. Clinical manifestations, Oregon-Green Dextran (OGD) staining, the apical junction complexes (AJCs), IL-33/ST2 and IL-9/IL-9R signaling molecules were evaluated in ocular surface and its draining cervical lymph nodes (CLNs) by RT-qPCR, immunostaining and ELISA. The typical allergic signs, enhanced OGD staining intensity, disrupted morphology of AJCs, including ZO-1, claudin 1, occludin, and E-cadherin, and the stimulated signaling of IL-33/ST2 and IL-9/IL-9R were observed in ocular mucosa and draining CLNs in EAC-Balb/c mice, but significantly reduced or eliminated in EAC-ST2-/- mice. Topical challenge of IL-9 resulted in the obvious OGD staining and disrupted ocular surface AJCs in Balb/c mice and in HCECs in vitro. IL-9 production was found to be stimulated by IL-33 in CD4+ cells from Balb/c mice in vitro. Our findings uncovered a novel phenomenon and mechanism by which ocular surface barrier integrity is disrupted in allergic conjunctivitis by IL-33/ST2/IL-9/IL-9R signaling pathway, which may amplify the allergic inflammation.