Estrogen has been reported to prevent development of cardiac hypertrophy in female rodent models and in humans. However, the mechanisms of sex steroid action are incompletely understood. We determined the cellular effects by which 17--estradiol (E2) inhibits angiotensin II (Ang II)-induced cardiac hypertrophy in-vivo. Two weeks of angiotensin infusion in female mice resulted in marked hypertrophy of the left ventricle, exacerbated by the loss of ovarian steroid hormones from oophorectomy. Hypertrophy was 51% reversed by the administration of E2 (insertion of 0.1 mg/21 day-release tablets). The effects of E2 were mainly mediated by the estrogen receptor (ER) beta isoform, since E2 had little effect in ER null mice, but comparably inhibited AngII-induced hypertrophy in wild type or ER null mice. AngII induced a switch of myosin heavy chain form production from to , but this was inhibited by E2 via ER. Ang II-induced ERK activation was also inhibited by E2 through the beta receptor. E2 stimulated brain natriuretic peptide protein expression, and substantially prevented ventricular interstitial cardiac fibrosis (collagen deposition) as induced by AngII. Importantly, E2 inhibited calcineurin activity that was stimulated by AngII, related to E2 stimulating the modulatory calcineurin-interacting protein (MCIP) 1 gene and protein expression. E2 acting mainly through ER mitigates the important signaling by AngII that produces cardiac hypertrophy and fibrosis in female mice.