Human Immunodeficiency virus type 1 (HIV-1) disproportionately affects women, accounting for >50% of new HIV infections in adults worldwide. While multiple mechanisms may contribute to a greater degree of HIV infection in women than men, we evaluated the direct effect of 17β-estradiol, the most bioactive form of estrogen in women, on HIV replication in peripheral blood mononuclear cells (PBMCs). We demonstrate that 17β-estradiol, in an ERα dependent manner, inhibits HIV replication by activating β-catenin signaling. Specifically, we show for the first time that 17β-estradiol induces a complex formation between ERα and β-catenin which tether on the HIV LTR at −143 nt site from +1 start site of HIV transcription to repress HIV promoter activity. These studies define a role of 17β-estradiol in inhibiting HIV replication which may impact HIV pathogenesis in women and add to a growing list of viruses that are inhibited by 17β-estradiol through ERα engagment.