Abstract Introduction and Objectives: Cancer stem-like cells (CSCs) represent a small subpopulation of largely quiescent cells that reside within tumors. Several studies have demonstrated that this population is more resistant to current therapies and is therefore directly responsible for tumor recurrence. Recent evidence suggests that the transcription factor STAT3 is crucial to the survival of stem cells in prostate cancer (PCa). Thus, inhibition of activated STAT3 (pSTAT3) is a valid strategy to selectively target PCa CSCs. We have previously shown that pSTAT3 blockade, by galielallactone (GL), not only reduces proliferation and induces apoptosis of prostate cancer cells in vitro, but it also inhibits the growth of prostate tumors and the metastatic spread to regional and distal lymph nodes, in vivo. Here we performed experiments aimed at studying the effect of the STAT3 inhibitor, GL, on PCa CSCs, as a promising therapeutic approach for prostate cancer patients. Materials and Methods: The expression of stem, basal, and luminal cell surface markers (CD133/CD44/CD24) was analyzed by FACS on DU145 and PC3 cells following treatment with GL. DU145 cells were sorted, based on the expression of the stem cell surface marker CD133, and then plated for further analysis. Expression of pSTAT3 was studied by IHC, on sorted cells, and the effect of GL on clonogenic recovery was determined. Furthermore, the effect of GL on sphere formation efficiency was studied on the CD133 population. The compound galiellalactone was obtained from Glactone Pharma (Sweden). Results: Our results show that the CSCs population (CD133+/CD44+) expresses high levels of activated pSTAT3 compared to the CD44+/CD24+ cell population. Treatment with GL decreased the number of CSCs in DU145 cells after 48h, while the number of CD44+/CD24+ cells was not significantly affected. Importantly, treatment with GL had no effect on CD133+/CD44+ or CD44+/CD24+ populations in PC3 cells, which do not express pSTAT3. Treatment with GL significantly reduced the ability of CSCs to form colonies and to generate prostate-derived spheres. Interestingly, the CSCs population (CD133+) was more sensitive to GL in terms of colony- and sphere-forming efficiency compared to the CD133- population. Conclusion: This study demonstrates that the STAT3 inhibitor galiellalactone can specifically target the prostate cancer stem-like cell population in vitro, implying that pSTAT3 inhibition by GL could represent a valid therapeutic strategy to antagonize CSCs in human prostate cancer. Future experiments will be aimed at validating these data and relating the clonogenic inhibition to the effects of pSTAT3 blockade by GL on tumor initiation by prostate cancer stem-like cells in vivo. Citation Format: Giacomo Canesin, Anne T. Collins, Rebecka Hellsten, Norman J. Maitland, Anders Bjartell. Targeting the prostate cancer stem cell niche with the STAT3 inhibitor galiellalactone [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A040.