Few studies have referred to the implication of anoikis processes following hormonal treatment. No data are available on the influence of estrogen in ovarian cancer anoikis. To gain insights into the effects and mechanism of estrogen in ovarian cancer cells, we have carried out studies on the anoikis of ovarian cancer cells treated with estrogen and on the pathways involved. We observed an anti-anoikis role of E2 in suspended Caov-3 cells, and this was mainly due to the decreasing of Bit1 level in cytosol. We also found that estrogen receptor α (ERα) was the main mediator involved in this process. To study the signaling pathways well, phosphatidylinositol 3-kinase (PI3K)/AKT were further investigated. Results demonstrated that the decreasing of the Bit1 level in cytosol mediated by E2 binding to ERα was mainly through PI3K/AKT pathways. Overall, these findings disclose a new perspective for estrogen on ovarian cancer therapy.