Radiotherapy is a curative treatment option in prostate cancer. Nevertheless, patients with high-risk prostate cancer are prone to relapse. Identification of the predictive biomarkers and molecular mechanisms of radioresistance bears promise to improve cancer therapies. In this study, we show that aldehyde dehydrogenase (ALDH) activity is indicative of radioresistant prostate progenitor cells with an enhanced DNA repair capacity and activation of epithelial-mesenchymal transition (EMT). Gene expression profiling of prostate cancer cells, their radioresistant derivatives, ALDH(+) and ALDH(-) cell populations revealed the mechanisms, which link tumor progenitors to radioresistance, including activation of the WNT/beta-catenin signaling pathway. We found that expression of the ALDH1A1 gene is regulated by the WNT signaling pathway and co-occurs with expression of beta-catenin in prostate tumor specimens. Inhibition of the WNT pathway led to a decrease in ALDH(+) tumor progenitor population and to radiosensitization of cancer cells. Taken together, our results indicate that ALDH(+) cells contribute to tumor radioresistance and their molecular targeting may enhance the effectiveness of radiotherapy. Cancer Res; 75(7); 1482-94. (c)2015 AACR.