Assisted reproductive technology (ART) has been widely used among women with infertility. However, the association of ART with birth defects and stillbirth remains controversial and has rarely been reported in China. A retrospective cohort study of 112,043 pregnant women and 114,522 newborns from 2006 to 2016 was performed. Compared to spontaneously conceived infants, ART-conceived infants had a higher likelihood of any birth defect, with an adjusted odds ratio (OR) of 2.10 (95% confidence interval, 1.63-2.69). ART-conceived infants also had a significantly increased risk for subcategories of cardiovascular, musculoskeletal, urogenital, gastrointestinal, and respiratory defects. Most (62.25%) of the effect of ART on birth defects was a direct effect, whereas 37.75% of the effect of ART on birth defects was due to multiple pregnancies (i.e., an indirect effect). Compared with naturally conceived singletons, the combined effect of ART and twins on the risk of birth defects was lower than that of the sum of the individual effects of ART and twins on the risk of birth defects, with an adjusted OR of 0.54 (0.32-0.92). These findings clearly show that ART is associated with an increased risk of birth defects in China and may provide guidance to couples and obstetricians in selecting numbers of pregnancies and in identifying organs at a high risk of birth defects.