Systemic glucocorticoid dexamethasone has known for exerting an inhibitory effect on tracheal mucin secretion and therefore considered the primary option for treating acute asthma exacerbation. However, the mechanism underlying glucocorticoid-induced decreased in mucosubstances is unclear. Recent studies have reported that dexamethasone exerts an inhibition on mucin and mucosubstances in the lung by modulating the expression of calcium-processing genes. However, the expression of the calcium-processing genes in trachea are not examined yet. Thus, the present study is the first to report glucocorticoid-induced regulation of tracheal calcium processing genes such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k), and plasma membrane Ca2+-ATPase (Pmca1) in the mice. In the study, mice were subcutaneously injected with systemic dexamethasone for 5days, or injected with estradiol or progesterone for 3 days. The tracheae were collected by dividing them into cervical and thoracic sections based on its anatomical structure. Quantitative PCR was performed to investigate mRNA expression of calcium-processing genes. Immunohistochemistry and immunofluorescence were performed to localize the calcium-processing proteins. Tracheal mucins were detected by performing Alcian blue-periodic acid-Schiff staining. The expression of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was localized in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment decreased the mRNA expression of the four calcium-processing genes and mucin 1, mucin 4, mucin 5ac, and mucin 5b genes. Dexamethasone inhibited in the secretion of mucosubstances in the trachea. Our findings suggest that glucocorticoids regulate the tracheal expression of calcium-processing genes and tracheal mucin secretion.