Abstract Cancer stem cells (CSCs) represent the most tumorigenic, metastatic and therapy resistant cell subpopulation within human tumors. Current therapies target bulk tumor cells while spare the most aggressive CSC subpopulation. Understanding the mechanisms responsible for the acquisition and maintenance of the CSC phenotype will help to identify new strategies to target CSCs. In this study, we describe a link between deregulated expression of the ETS transcription factor ESE3/EHF and upregulation of Lin28A and Lin28B in prostate CSC-enriched subpopulations. Furthermore, using various cell line models and in vitro/in vivo experimental systems we demonstrate the efficacy of targeting the Lin28A/B axis for selective elimination of cancer cells with tumor-initiating and stem-like properties. Mechanistically, we found that ESE3/EHF represses transcription of Lin28A and Lin28B in normal prostate epithelial cells. Downregulation of ESE3/EHF in prostate tumors led to upregulation of Lin28A and Lin28B and consequent reduction of microRNAs (miRNAs) of the let-7 family, which exert tumor suppressor functions. These events promoted cell transformation and expansion of the prostate CSC subpopulation. Conversely, targeting Lin28A/Lin28B with small interfering RNAs (siRNAs) in transformed prostate epithelial cells and prostate cancer cell lines restored the levels of let-7 miRNAs, decreased expression of several CSC marker genes, and restrained tumor-sphere formation and self-renewal properties in vitro and tumor-initiating capability in vivo. Notably, systemic treatment with a siRNA targeting Lin28B reduced growth of prostate tumor xenografts in mice. This was associated with a significant contraction of the CSC subpopulation in tumor xenografts, as demonstrated by the reduced content of ex vivo tumor-sphere forming cells, reduced expression of CSC marker genes, and upregulation of let-7 miRNAs. Furthermore, tumor cells derived from siLin28B-treated xenografts exhibited reduced in vivo tumor-initiating and self-renewal capability, in line with a persistent loss of CSC properties. Collectively, these data establish the Lin28/let-7 axis as a critical element in malignant transformation and acquisition of tumor-initiating and stem-like properties and identify a valid therapeutic strategy to antagonize CSCs in human prostate cancer. Citation Format: Domenico Albino, Gianluca Civenni, Cecilia Dallavalle, Martina Roos, Hartmut Jahns, Jonathan Hall, Giuseppina M. Carbone, Carlo V. Catapano. Targeting the Lin28A/B axis reverts stem cell-like phenotype and tumor-initiating properties in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3343.