ABSTRACT: BACKGROUND:China implemented a partial two-child policy (2013) followed by a universal two-child policy (2015), replacing the former one-child policy mandated by the government. The changes affect many aspects of China's population as well as maternal and infant health, but their potential impact on birth defects (BDs) remains unknown. In this study, we investigated the associations of these policy changes with BDs in Zhejiang Province, China. METHODS AND FINDINGS:We used data from the BD surveillance system in Zhejiang Province, China, which covers 90 hospitals in 30 urban districts and rural counties, capturing one-third of the total births in this province. To fully consider the time interval between conception and delivery, we defined the one-child policy period as data from 2013 (births from October 2012 to September 2013), the partial two-child policy period as data from 2015 (births from October 2014 to September 2015), and the universal two-child policy period as data from 2017 (births from October 2016 to September 2017). Data from 2009 and 2011 were also used to show the changes in the proportion of births to women with advanced maternal age (35 years and older) prior to the policy changes. Main outcome measures were changes in the proportion of mothers with advanced maternal age, prevalence of BDs, rankings of BD subtypes by prevalence, prenatal diagnosis rate, and live birth rate of BDs over time. A total of 1,260,684 births (including live births, early fetal losses, stillbirths, and early neonatal deaths) were included in the analyses. Of these, 644,973 (51.16%) births were to women from urban areas, and 615,711 (48.84%) births were to women from rural areas. In total, 135,543 (10.75%) births were to women with advanced maternal age. The proportion increased by 85.68%, from 8.52% in 2013 to 15.82% in 2017. However, it had remained stable prior to policy changes. Overall, 23,095 BDs were identified over the policy changes (2013-2017). The prevalence of BDs during 2013, 2015, and 2017 was 245.95, 264.86, and 304.36 per 10,000 births, respectively. Trisomy 21 and other chromosomal defects increased in both risk and ranking from 2013 to 2017 (crude odds ratio [95% confidence interval] 2.13 [1.75-2.60], from ranking 10th to 5th, and 3.63 [2.84-4.69], from ranking 16th to 6th, respectively). The prenatal diagnosis rate increased by 3.63 (2.2-5.1) percentage points (P < 0.001), from 31.10% to 34.72%, and identification of BDs occurred 1.88 (1.81-1.95) weeks earlier (P < 0.001). The live birth rate for infants with BDs born before 28 gestational weeks increased from 1.29% to 11.45%. The major limitations of this observational study include an inability to establish causality and the possible existence of unknown confounding factors, some of which could contribute to BDs. CONCLUSIONS:In this study, we observed significant increases in maternal age and the prevalence of total and age-related anomalies following China's new two-child policy. Increases in live birth rate for infants with BDs born before 28 gestational weeks suggest that healthcare for very preterm births with BDs may be warranted in the future, as well as updating the definition of perinatal period.