Background and objective: The repairing of the respiratory airway epithelium requires the exquisite coordination of progenitor proliferation and differentiation to achieve complex circuit assembly. Canonical Wnt/β-catenin signaling acts as an integrating molecule for multiple proliferation and differentiation signals of a variety of stem or progenitor cells. However, little is known about its role in repairing of respiratory airway epithelium.Methods: Using K5-CreERT2 transgenic mouse line for lineage tracing, we first show that airway basal cells (BCs) generate Clara and ciliated cells during naphthalane-induced airway injury. In vitro air-liquid interface (ALI) culture and clonal sphere-forming assay further revealed the involvement of Wnt signaling in self-renewal and multi-differentiation of BCs. Moreover, conditional deletion of β-catenin in mouse K5 BCs caused loss of BC proliferation and the repairing of Clara and ciliated cells.Results: Our results indicate that canonical Wnt/β-catenin signaling is crucial for the homeostatic maintenance function of airway BCs during postnatal injury. In vivo lineage tracing of mouse tracheal BCs, the majority 70% of lineage labeled cells were BCs and 30% of other labeled cells were Clara cells and ciliated cells at 6 weeks. Besides, tracheospheres with diameters ranging from 50μm to 200μm formed within 9 days and the percentage of K5 BCs was 90%. BCs treated in Wnt3A had an average of 125.5+4.6 (P＜0.00001) in the total cell number, the total K5-positive cell number was 100% (P＜0.001), the BrdU-positive ratio was 71.6+2.1 (P＜0.0001), and the BrdU-positive ratio of K5-positive cells was 86.0+2.6 (P＜0.0001) compared to the control, while those treated with DKK1 significantly decreased (P＜0.0001) in these indices compared to the control.Conclusions: Wnt/β-catenin was demonstrated as the signaling pathway of BC self-renewal and differentiation, which can be based to identify the etiologies of human abnormal airway remodeling in respiratory diseases and develop new strategies in treating these diseases.