Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.