Δ9-Tetrahydrocannabinol (Δ9-THC) has been reported as possessing antiestrogenic 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 showed that Δ9-THC exposures markedly suppresses 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 antiestrogenic activities are mediated by the ERβ disruption of E2/ERα signaling.