Epithelial cells play an important role in orchestrating mucosal immune responses. In allergic‐type inflammation, epithelial cells control the recruitment of eosinophils into the mucosa. Th2‐type cytokine‐driven release of eosinophil‐active chemokines from epithelial cells directs eosinophil migration into the mucosal epithelium. CCR3, the main eosinophil chemokine receptor, regulates this process; however, the respective contribution of individual CCR3 ligands in eosinophil transepithelial migration is less well understood. Using an in vitro transepithelial chemotaxis system, we found that eotaxin‐3 produced by IL‐4‐stimulated airway epithelial cells and CCR3 on eosinophils exclusively mediate eosinophil transepithelial migration. Eotaxin‐3 protein levels were also increased in the nasal mucosal epithelium recovered from allergic patients as compared to non‐allergic patients. Surprisingly, eotaxin‐3 in IL‐4‐stimulated airway epithelial cells was predominantly cell surface bound, and the cell surface form was critical for eosinophil transepithelial migration. Eotaxin‐3 cell surface association was partially glycosaminoglycan (GAG) dependent, but was completely protein dependent, suggesting that eotaxin‐3 associates with both GAG and cell surface proteins. We thus provide evidence that cell surface‐associated eotaxin‐3 is the critical IL‐4‐dependent chemotactic signal mediating eosinophil transepithelial migration in the setting of allergic inflammation.