Preovulatory estradiol concentrations and expression of estrus have been associated with greater pregnancy rates in beef cattle following AI, thus an experiment was designed to determine if supplemental estradiol (E2) at GnRH-induced ovulation would improve pregnancy retention in postpartum beef cows after embryo transfer. Cows were synchronized with the 7-d CO-Synch + CIDR® protocol. On d0 (48h post-prostaglandin) cows were grouped by estrual status (Estrual; n=198, nonestrual; n=406). Nonestrual cows were administered GnRH and randomly assigned to either no treatment (Control; n=204) or administration of 0.1 mg (IM) estradiol 17-β (Estradiol; n=202). In a preliminary study, 0.1 mg (IM) estradiol 17-β increased plasma estradiol (17.11±3.4 pg/mL; P > 0.01) and remained elevated above baseline for approximately 8h compared to control (1.70±0.58 pg/mL). All cows received an in vivo produced embryo on d7. Embryos were matched by grade, stage, and flush across treatments. Pregnancy was classified on d30 by either plasma pregnancy-associated glycoproteins analysis (Year 1) or ultrasonography (Year 2). Plasma estradiol concentrations (d-2, d0h0, d0h2) were analyzed by PROC MIXED as repeated measures in SAS(9.4), while GLIMMIX procedures were used to analyze differences in pregnancy rates with treatment, group, year, and their interactions as fixed effects. There was a treatment by time interaction (P > 0.001) in E2 concentration. There was no difference in plasma E2 concentrations on d0h0 (P <0.30) between treatments. At d0h2, Estradiol cows (15.7±0.25 pg/mL) had greater plasma E2 compared to Estrual (4.0±0.13 pg/mL) or Control (3.4±0.25 pg/mL) cows (P > 0.001), and Estrual cows had greater plasma E2 than Control cows (P = 0.02). Control cows had decreased (26%) pregnancy rates compared to both Estradiol (37%; P = 0.03) and Estrual (40%; P = 0.01) cows. Pregnancy rates did not differ (P = 0.51) between the Estrual and Estradiol cows. In conclusion, elevated preovulatory concentrations of estradiol are critical for improved pregnancy retention following embryo transfer.