Objectives: Obesity is associated with increased risk of and mortality from endometrial cancer (EC). Previous studies have shown that omega-3 polyunsaturated fatty acid (PUFA) supplementation may counteract the detrimental effects of obesity-driven cancers, but this has not been thoroughly investigated in EC. We aimed to assess the effect of omega-3 PUFAs on cell proliferation and tumor growth in endometroid EC cell lines and an LKB1fl/flp53fl/fl mouse model of endometroid EC. Methods: The human endometrioid EC cell lines ECC-1 and KLE were exposed to varying concentrations of the omega-3 PUFA, docosahexaenoic acid (DHA). Cell proliferation was assessed by MTT assay. Apoptosis was assessed by Annexin V-FITC assay. Reactive oxygen species (ROS) were measured using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Western immunoblotting was performed to determine the effects of DHA on the anti-apoptotic proteins BCL-2 and MCL-1 and the cellular stress proteins PERK, Bip, and PDI. LKB1fl/flp53fl/fl mice were fed either a low fat diet (10% of calories from fat, lean) or high fat diet (60% of calories from fat, obese). Following EC onset, the mice were treated with placebo or DHA (15mg/kg weekly for 4 weeks, IP). Immunohistochemistry (IHC) was performed on tumors in the treated and untreated mice to assess for proliferation and apoptosis. Global, unbiased metabolomics was used to identify the metabolic effects of DHA in the endometrial tumors. Results: DHA inhibited cell proliferation in a dose-dependent manner in both cell lines (ECC-1 IC50 of 60µM; KLE IC50 of 35µM). DHA increased the expression of annexin V (pConclusions: DHA robustly inhibits EC proliferation and tumor growth in vitro and in vivo. This suggests that omega-3 PUFA supplementation may be a promising dietary strategy for EC prevention and treatment. Citation Format: Jillian A. O'Donnell, Lindsey K. Buckingham, Stuart R. Pierce, Lindsay West, Yiajie Yin, Wenchuan Sun, Ziwei Fang, Douglas P. Lee, Chunxiao Zhou, Victoria L. Bae-Jump. Docosahexaenoic acid demonstrates anti-tumorigenic effects in endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO031.