Estrogen is an important regulator of dermal fibroblast functions, including extracellular matrix (ECM) synthesis. Estrogen mediates its effects through estrogen receptors (ERs), ERα and ERβ; however, regulation of ERs in dermal fibroblasts remains poorly understood. Friend leukemia integration factor 1 (Fli1), a member of the Ets transcription factor family, has been shown to play a pivotal role in regulation of the ECM genes in dermal fibroblasts. The aim of this study was to examine a possible interaction between Fli1 and estrogen pathways, focusing on ERα. We show that treatment of human dermal fibroblasts with transforming growth factor-β (TGF-β) increases ERα protein and mRNA levels. Similarly, ERα expression was increased in response to small interfering RNA (siRNA)-mediated depletion of Fli1, suggesting that Fli1 is a mediator of the TGF-β effects on ERα expression. Accordingly, we showed that Fli1 binds to the most proximal region of the ERα promoter, and dissociates from the promoter upon TGF-β treatment. An inverse correlation between Fli1 and ERα expression levels was confirmed in cultured skin fibroblasts obtained from Fli1(+/-) mice and in the skin of Fli1(+/-) mice in vivo. This study supports a role of Fli1 as a negative regulator of the ERα gene in dermal fibroblasts.