Objective To investigate the epidemiological changes in extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) vaginal colonization in high-risk pregnant women and identify independent risk factors. Further, the differences in perinatal outcomes according to maternal ESBL-E vaginal colonization were analyzed. Design Retrospective cohort study. Setting Republic of Korea Population 1,460 women admitted to our high-risk pregnancy unit between 14+0 and 35+6 gestational weeks. Methods The study period was divided into periods 1 (January 2010 to July 2015) and 2 (August 2015 to December 2020). The main outcomes were analyzed according to each period and ESBL-E vaginal colonization. Main Outcome Measures ESBL-E vaginal colonization rate, risk factors for ESBL-E vaginal colonization, and perinatal outcomes. Results The ESBL-E vaginal colonization rate was higher in period 2, which was attributed to a significantly higher proportion of ESBL-producing Escherichia coli. Cerclage (odds ratio [OR]: 3.248; 95% confidence interval [CI]: 1.744–6.049) and prior antibiotic treatment (OR: 3.044; 95% CI: 1.713–5.410) were found as independent risk factors for ESBL-E vaginal colonization. Earlier gestational age at delivery, and higher proven early-onset neonatal sepsis (EONS) rate were observed in the ESBL-E-positive group. Conclusions The ESBL-E vaginal colonization rate in high-risk pregnant patients has increased over the past decade, and the independent risk factors for colonization are cerclage and prior antibiotic treatment. Additionally, maternal ESBL-E vaginal colonization is associated with higher proven EONS rates. Funding This study received no funding. Keywords Extended-spectrum β-lactamase, Enterobacteriaceae, vaginal colonization, antibiotics use, cerclage, neonatal sepsis