Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) has been reported as possessing anti-estrogenic activity, although the mechanisms underlying these effects are poorly delineated. In this study, we used the estrogen receptor α (ERα)-positive human breast cancer cell line, MCF-7, as an experimental model and show that Δ⁹-THC exposures markedly suppress 17β-estradiol (E2)- induced MCF-7 cell proliferation. We demonstrate that these effects result from Δ⁹-THC’s ability to inhibit E2-liganded ERα activation. Mechanistically, the data obtained from biochemical analyses revealed that: i) Δ⁹-THC up-regulates ERβ, a repressor of ERα, inhibiting the expression of E2/ERα-regulated genes that promote cell growth, and that ii) Δ⁹-THC induction of ERβ modulates E2/ERα signaling in the absence of direct interaction with the E2 ligand binding site. Therefore, the data presented support the concept that Δ⁹-THC’s anti-estrogenic activities are mediated by the ERβ disruption of E2/ERα signaling. αααααααααα