Abstract Background: Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized that the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions. Methods: The increase of pSTAT3 Tyr705 in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens was determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies of pSTAT3 Tyr705 were used to demonstrate the necessity for cell survival and proliferation under hypoxic conditions. In addition, S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). This association was evaluated with knock down of S1PR1 in HOCCs. HOCCs were treated with our novel STAT3 inhibitor, HO-3867. Results: pSTAT3 Tyr705 is increased in the hypoxic regions of EOC specimens. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. There was decreased expression of the STAT3-target proteins Bcl-xL, cyclin D2, and VEGF in all HO-3867 treated cells. Conclusions: These findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Therefore, targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors. Citation Format: Georgia A McCann, MD, Adam ElNaggar, MD, Shan Naidu, MS; Kellie S Rath, MD; Brent J Tierney, MD; Adrian Suarez, MD; David E Cohn, MD; Karuppaiyah Selvendiran, PhD. Targeting constitutively-activated STAT3 in hypoxic ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1413.