Although involved in processes leading to the emergence and development of hormone-dependent breast cancers, the estrogen receptor a (ERα) also prevents transformed cells from progressing toward a more aggressive phenotype. The transcriptional activity of ERα is mediated through two transactivation functions, called activation function 1 and 2, whose respective involvement varies in a cell-specific manner. Here, we identify the Rho/megakaryoblastic leukemia 1 (MKL1) signaling pathway as a main actor in controlling the cell-specific activity of both transactivation functions of ERα. Notably, we show that, when the coregulator MKL1 is sequestered in an inactive form by unpolymerized actin, the transcriptional activity of ERα mainly relies on the activation function 1. The activation of MKL1, which results from its dissociation from unpolymerized actin, promoted by the ability of Rho to support polymeric actin accumulation, silences the activation function 1 of ERα and allows the receptor to mainly act through its activation function 2. Importantly, this switch in the respective contribution exerted by both transactivation functions is correlated with an impaired ability of ERα to efficiently transactivate estrogen-regulated reporter genes. MKL1 is further shown to be present on estrogen-responsive genes in vivo. Interestingly, the Rho/MKL1 signaling pathway is activated during the epithelial-mesenchymal transition. A reduced transactivation efficiency of ERα, resulting from the activation of this pathway, may therefore suppress the protective role exerted by ERα toward tumor progression and invasiveness. [ABSTRACT FROM AUTHOR]