In January 2000, Chilean Ministry of Health mandated the addition of folic acid (FA) to wheat flour in order to reduce the risk of neural tube defects (NTDs). This policy resulted in significant increases in serum and red cell folate in women of fertile age 1 year after fortification. To evaluate the effect of wheat flour fortification on the prevalence of NTDs in Chile we designed a prospective hospital-based surveillance program to monitor the frequency of NTDs in all births (live and stillbirths) with birth weight ≥500 g at the nine public maternity hospitals of Santiago, Chile from 1999 to 2009. During the pre-fortification period (1999–2000) the NTD rate was 17.1/10,000 births in a total of 120,566 newborns. During the post-fortification period (2001–2009) the NTD rate decreased to 8.6/10,000 births in a total of 489,915 newborns, which translates into a rate reduction of 50% (RR: 0.5; 95% CI: 0.42–0.59) for all NTDs. The rate reduction by type of NTD studied was: 50% in anencephaly (RR: 0.5; 95% CI: 0.38–0.67), 42% in cephalocele (RR: 0.58; 95% CI: 0.37–0.89), and 52% in spina bifida (RR: 0.48; 95% CI: 0.38–0.6). Rates showed significant reduction both in stillbirths and live births: 510.3 to 183.6/10,000 (RR = 0.36; 95% CI: 0.25–0.53) and 13.3 to 7.5/10,000 (RR = 0.56; 95% CI: 0.47–0.68), respectively. In Chile, fortification of wheat flour with FA has proven to be an effective strategy for the primary prevention of NTDs. © 2012 Wiley Periodicals, Inc.