Metastatic prostate cancer represents a yet unsolved clinical problem due to the high frequency of relapse and treatment resistance. Understanding the pathways that lead to prostate cancer progression is an important task to prevent this deadly disease. The ETS transcription factor ESE3/EHF has an important role in differentiation of human prostate epithelial cells. Loss of ESE3/EHF in prostate epithelial cells determines transformation, epithelial-to-mesenchymal transition (EMT) and acquisition of stem-like properties. In this study we identify IL-6 as a direct target of ESE3/EHF that is activated in prostate epithelial cells upon loss of ESE3/EHF. ESE3/EHF and IL-6 were significantly inversely correlated in prostate tumors. Chromatin immunoprecipitation confirmed binding of ESE3/EHF to a novel ETS binding site in the IL-6 gene promoter. Inhibition of IL-6 reverted transformation and stem-like phenotype in tumorigenic ESE3/EHF knockdown prostate epithelial cell models. Conversely, IL-6 stimulation induced malignant phenotypes, stem-like behavior and STAT3 activation. Increased level of IL-6 was observed in prostatospheres compared with adherent bulk cancer cells and this was associated with stronger activation of STAT3. Human prostate tumors with IL-6 elevation and loss of ESE3/EHF were associated with STAT3 activation and displayed upregulation of genes related to cell adhesion, cancer stem-like and metastatic spread. Pharmacological inhibition of IL-6/STAT3 activation by a JAK inhibitor restrained cancer stem cell growth in vitro and inhibited self-renewal in vivo. This study identifies a novel connection between the transcription factor ESE3/EHF and the IL-6/JAK/STAT3 pathway and suggests that targeting this axis might be preferentially beneficial in tumors with loss of ESE3/EHF.