Klotho is a recently discovered antiaging protein. Although many researchers are investigating the roles of Klotho in chronic kidney diseases and cancer, however, there are no studies on the roles of Klotho in chronic pulmonary diseases. The purpose of this study was to define the role of Klotho in pulmonary fibrosis using a murine model of ovalbumin (OVA)-induced chronic asthma and in BEAS-2B human bronchial epithelial cells. In an in vivo experiment, mice were sensitized by intraperitoneal injection of OVA (20 μg/mouse), followed 1 week later by an airway challenge with 1 % OVA solution delivered three times a week for 4 weeks. In an in vitro experiment, we investigated the effects of stimulated with interleukin (IL)-4 and tumor necrosis factor (TNF)-α on Klotho protein and VEGF and transforming growth factor (TGF)-β1/Smad3 signaling in BEAS-2B cells. Klotho decreased and VEGF and TGF-β1 levels increased with increasing duration of OVA challenge. Similar findings were found for the expression of these proteins in lung tissue. The collagen content in lung tissue increased with repeated OVA challenge. In the in vitro experiment, Klotho expression decreased and VEGF and TGF-β1/Smad3 expression increased after IL-4 (50 ng/mL) and TNF-α (50 ng/mL) stimulation. Pretreatment with 25, 50, and 100 ng/mL of Klotho protein significantly attenuated the increases in VEGF and TGF-β1/Smad3 expression levels after IL-4 and TNF-α treatment, and reduced α-smooth muscle actin expression in concentration-dependent manner. Klotho protein inhibited the fibrotic response by suppressing VEGF and TGF-β1/Smad3 expression. These results suggest that Klotho protein may be crucial to inhibiting fibrosis associated with chronic airway diseases.