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