Allergen-derived proteases are critical in asthma development. House dust mite (HDM) cysteine protease activity disrupts the airway epithelial barrier and is inhibited by cystatin SN (CST1). We aimed to elucidate the role of epithelium-derived CST1 in HDM-induced asthma. CST1 protein in human lung tissue was determined by immunohistochemistry (n=9 healthy controls, n=8 asthma). Sputum and serum CST1 protein levels were measured by ELISA. CST1 suppression of HDM-induced bronchial epithelial barrier function was examined in vitro in 16HBE cells and in vivo in a mouse model. CST1 protein expression was higher in the bronchial epithelium of asthmatics than in healthy controls in lung tissues (p=0.0003). IL-13 significantly both upregulated CST1 mRNA and protein levels in 16HBE cells in concentration dependent manner. Induced sputum CST1 levels were higher in 83 asthmatics than in 21 control subjects (142±9 vs 39±7 ng/ml, p<0.0001) independent of asthma control status. Serum CST1 protein levels were higher in 59 asthmatics than in 17 controls (1129±74 vs 703±57 pg/ml, p<0.004). Sputum and serum CST1 protein levels were negatively correlated with FEV1, FEV1%pred and FEV1/FVC; and positively correlated with IL-13 protein levels (sputum: r=0.42, p=0.008; serum: r=0.38, p=0.01) in asthma. Serum CST1 protein levels negatively correlated with HDM-specific IgE (sIgE) protein levels in asthmatics (r=-0.48, p=0.002). HDM-induced epithelial barrier function disruption was suppressed by recombinant human CST1 protein (rhCST1) in vitro and in vivo. We conclude that human CST1 suppresses asthma symptoms by protecting the asthmatic bronchial epithelial barrier through inhibiting allergen protease activity.