Display omitted There is increasing evidence for sexual dimorphism of estrogen (E2) actions in the exacerbation of lung function, infection and inflammation in females with cystic fibrosis - the so-called “CF gender gap”. The effects of estrogen on virulence factors that enhance P. aeruginosa persistence in CF lung epithelium were investigated by phenotypic and chemical assays in various PsA clinical isolates and laboratory strains in isolation or in co-culture with normal (Nuli-1) and CF dPhe508-CFTR (CuFi-1) human bronchial epithelial cell lines. Estrogen (E2, 10 nM) significantly increased secretion of the virulence factor pyocyanin by 80% in PsA early infection isolates from female CF patients and by 280% in late infection PsA isolates. Estrogen also increased the swarming motility by up to 50% in all PsA isolates and strains tested in 0.5% agar. A significant increase of 110% in the twitching motility of all PsA isolates and strains tested was also observed with estrogen treatment. Treatment with E2 increased biofilm formation of P. aeruginosa PsAO1 which became more adherent to, and invasive into, normal and CF bronchial epithelial cells. The selective estrogen receptor modulators (SERMs), Tamoxifen and ICI 182780 inhibited P. aeruginosa motility. The potency of various steroid hormones to stimulate motility of P. aeruginosa was in the order; estradiol ≫ estrone > E3 estriol ≥ testosterone ≥ progesterone ≫ aldosterone, cortisol. Estrogen was also shown to reduce ciliary beat intensity in CF bronchial epithelium which would further exacerbate PsA trapping and virulence in the CF airways. In conclusion, we have demonstrated for the first time that estrogen exacerbates P. aeruginosa virulence factors and enhances bacterial interactions with CF bronchial epithelium which can be inhibited by tamoxifen. Our work suggests that SERMs could be used as an adjuvant treatment to reduce estrogen-induced P. aeruginosa infections and associated lung exacerbations in females with CF.