Expression of the transcriptional regulator, E26 transformation-specific 1 (ETS1), is elevated in human prostate cancers, and this is associated with more aggressive tumor behavior and a rapid progression to castrate-resistant disease. Multiple ETS1 isoforms with distinct biological activities have been characterized and in 44 matched nonmalignant and malignant human prostate specimens, messenger RNAs for two ETS1 isoforms, ETS1p51 and ETS1p42, were detected, with ETS1p51 levels significantly lower in prostate tumor compared to matched nonmalignant prostate tissues. In contrast, ETS1p51 protein, the only ETS1 isoform detected, was expressed at significantly higher levels in malignant prostate. Analysis of epithelial-to-mesenchymal transition (EMT)–associated genes regulated following overexpression of ETS1p51 in the LNCaP prostate cancer cell line predicted promotion of transforming growth factor β (TGFβ) signaling and of EMT. ETS1p51 overexpression upregulated cellular levels of the EMT transcriptional regulators, ZEB1 and SNAIL1, resulted in reduced expression of the mesenchymal marker vimentin with concomitantly elevated levels of claudin 1, an epithelial tight junction protein, and increased prostate cancer cell migration and invasion. ETS1p51-induced activation of the pro-EMT TGFβ signaling pathway that was predicted in polymerase chain reaction arrays was verified by demonstration of elevated SMAD2 phosphorylation following ETS1p51 overexpression. Attenuation of ETS1p51 effects on prostate cancer cell migration and invasion by inhibition of TGFβ pathway signaling indicated that ETS1p51 effects were in part mediated by induction of TGFβ signaling. Thus, overexpression of ETS1p51, the predominant ETS1 isoform expressed in prostate tumors, promotes an EMT program in prostate cancer cells in part via activation of TGFβ signaling, potentially accounting for the poor prognosis of ETS1-overexpressing prostate tumors.