Objectives: To evaluate the relationship between peak estradiol levels on the ovulation trigger day and reproductive outcomes in IUI cycles. Design: A retrospective cohort study. Setting: China. Sample: The cohort consisted of 7525 IUI cycles during the period 2019-2023. Methods: We used data from three institutions. Outcomes included live birth, clinical pregnancy, miscarriage, ectopic pregnancy, multiple pregnancy, preterm birth and neonatal growth parameters. Statistical analyses, including multivariate stepwise regression and mediation analysis, were conducted to evaluate estradiol’s impact on outcomes. Main Outcome Measures: The miscarriage rate and live birth rate with low peak estradiol levels. Results: The highest estradiol quartile exhibited higher live birth (13.5%, P < 0.001) and clinical pregnancy rates (17.3%, P = 0.004). Conversely, the lowest estradiol quartile had increased miscarriage risk (OR = 2.15, P = 0.012) and reduced live birth rates (OR = 0.60, P = 0.070). Specifically, non-letrozole cycles increased miscarriage risk (OR = 4.30, P = 0.005) and decreased live birth rates (OR = 0.31, P = 0.010), whereas no significant impact was observed in letrozole cycles. Estradiol significantly mediated the effect of ovulation stimulation type on reproductive outcomes, with a suppression effect observed (PM = 180.5%). Funding: Medical Research Fund of Guangdong Province (A2024003), and Xinjiang Support Rural Science and Technology Program in Guangdong Province (KTPYJ 2023014). Conclusions: Higher estradiol levels on the ovulation trigger day enhance live birth and clinical pregnancy rates. Low estradiol levels, especially in non-letrozole cycles, are associated with higher miscarriage risks and poorer reproductive outcomes.