In the present report, we investigated the action of retinoic acid (RA) on the transactivation of the epidermal growth factor receptor (EGFR) gene promoter. In a previous study, we showed that the estrogen receptor (ER) ⍺ activated by 17β-estradiol (E₂) increased EGFR expression by enhancing the binding of the transcription factor Sp1 to the EGFR minimal promoter in HeLa cells. Here, we demonstrate that ligand-activated RA receptor (RAR) ⍺ inhibited EGFR transactivation by competing with Sp1 for binding to the same promoter fragment in the same cell model. When RAR⍺ and ER⍺ were coexpressed, the inhibitory effect of RA on transactivation of the EGFR promoter counteracted the enhancement induced by E₂-activated ER⍺ and became more pronounced in the presence of ligand-free ER⍺. In the MCF7 breast cancer cell line, which endogenously expresses RAR⍺ and ER⍺, RA exerted anti-proliferative effects in the presence of ligand-free ER⍺. Moreover, interplay between the pathways mediated by the two receptors was observed, as RA counteracted E₂-induced cell proliferation. Our results suggest that the interference with the activity of Sp1 on the EGFR promoter could be related to the observed RA-mediated growth suppression of breast cancer cells.