Background: Regular aspirin use may lower ovarian cancer risk by blocking the cyclooxygenase enzymes, resulting in lower expression of prostaglandins, including prostaglandin E2 (PGE2). We evaluated whether higher prediagnosis PGE-M (a urinary biomarker of PGE2) was associated with increased ovarian cancer risk in three prospective cohorts. Methods: We conducted a case–control study nested in the Nurses' Health Study (NHS), NHSII, and Shanghai Women's Health Study. Our analyses included 304 cases of epithelial ovarian cancer diagnosed from 1996 to 2015 and 600 matched controls. We measured urinary PGE-M using LC/MS with normalization to creatinine. Measures from each study were recalibrated to a common standard. We estimated ORs and 95% confidence intervals (CI) using conditional logistic regression, with PGE-M levels modeled in quartiles. Multivariable models were adjusted for ovarian cancer risk factors. Results: There was no evidence of an association between urinary PGE-M levels and ovarian cancer risk for women with PGE-M levels in the top versus bottom quartile (OR ¼ 0.80; 95% CI, 0.51–1.27; Ptrend ¼ 0.37). We did not observe heterogeneity by histotype (P ¼ 0.53), and there was no evidence of effect modification by body mass index (Pinteraction ¼ 0.82), aspirin use (Pinteraction ¼ 0.59), or smoking (Pinteraction ¼ 0.14). Conclusions: Prediagnosis urinary PGE-M levels were not significantly associated with ovarian cancer risk. Larger sample sizes are needed to consider a more modest association and to evaluate associations for specific tumor subtypes. Impact: Systemic prostaglandin levels do not appear strongly associated with ovarian cancer risk. Future research into aspirin use and ovarian cancer risk should consider local prostaglandins and prostaglandin-independent mechanisms.