The initiating events of ovarian cancer remain unknown, but an established risk factor is use of estrogen therapy by post-menopausal women where there is a positive correlation between duration of use and risk for disease. Mouse models of ovarian cancer have shown that exposure to exogenous 17β-estradiol (E2) accelerates tumour onset so this study aims to investigate the E2 signalling mechanisms responsible for sensitizing ovarian epithelial cells to transformation. By developing model systems that are responsive to E2 manipulation, we showed that E2 induces the formation of epithelial dysplasias both in vitro and in vivo. microRNA microarray was used to discover that E2 up-regulates microRNA-378 via the ESR1 pathway, resulting in the down-regulation of a tumour suppressor gene called Disabled-2 (Dab2). E2 suppression of Dab2 was found to result in increased proliferation, loss of contact inhibition, epithelial dysplasia, and increased sensitivity to transformation. This mechanism was also found to be active in mouse fallopian tube epithelium and human ovarian cancer cells. Single-cell RNA sequencing and trajectory analysis was subsequently used to explore additional signalling mechanisms that might contribute to the emergence of dysplastic lesions induced by E2. Multiple molecular signalling pathways dysregulated by E2 were identified and this revealed several possible biomarkers to be investigated for early detection of ovarian cancer. In the context of a current lack of strategies for ovarian cancer prevention or early detection, this work represents a significant advance in our understanding of how E2 promotes ovarian cancer initiation.