ImportanceHalf of women who are postmenopausal have genitourinary discomfort after menopause. Recommended therapies include low-dose vaginal estrogen. Individuals with a history of breast cancer or venous thromboembolism may have concerns about the safety of this intervention.ObjectiveTo compare serum estrogen concentrations with the use of vaginal estrogen, 10 μg, tablet vs placebo in women who are postmenopausal.Design, Setting, and ParticipantsThis is a secondary, post hoc analysis of data from a randomized clinical trial of treatment for moderate to severe genitourinary syndrome in women who are postmenopausal. The study was conducted at Kaiser Permanente Washington Health Research Institute and the University of Minnesota from April 11, 2016, to April 23, 2017. Measurements and data analysis were performed from November 3, 2020, to September 23, 2022.InterventionsParticipants were randomly assigned to vaginal estradiol tablet (10 μg/d for 2 weeks and then twice weekly) plus placebo gel (3 times weekly) or dual placebo for 12 weeks.Main Outcomes and MeasuresIn this post hoc analysis, baseline and week 12 serum estradiol, estrone, and sex hormone–binding globulin (SHBG) concentrations were measured by a chemiluminescent assay. Week 12 values of the 3 analytes were compared by baseline participant characteristics. Linear models compared week 12 estradiol concentrations between treatment groups, adjusted for baseline characteristics.ResultsA total of 174 women, mean (SD) age 61 (4) years, were included. Those in the estrogen group (n = 88) were more likely to have higher geometric mean (SD) week 12 serum estradiol concentrations (4.3 [2.2 pg/mL]) than those in the placebo group (n = 86) (3.5 [2.1] pg/mL) (P = .01). Adjusted for pretreatment hormone concentrations, age, clinical site, and body mass index, assignment to the estrogen vs placebo treatment group was significantly associated with higher week 12 estradiol concentrations (23.8% difference; 95% CI, 6.9%-43.3%). Most (121 of 174 [69.5%]) participants had enrollment serum estradiol concentrations higher than 2.7 pg/mL. Of women starting treatment at estradiol levels lower than or equal to 2.7 pg/mL, 38.1% (8 of 21) in the estrogen group and 34.4% (11 of 32) in the placebo group had estradiol concentrations higher than 2.7 pg/mL after 12 weeks of study participation (P = .78). Treatment assignment was not associated with week 12 estrone or SHBG concentrations.Conclusions and RelevanceIn this secondary analysis of a randomized clinical trial, a significant, although small, increase in serum estradiol levels was noted after 12 weeks of vaginal estrogen administration. The clinical relevance of this small increase is uncertain.Trial RegistrationClinicalTrials.gov Identifier: NCT02516202