Aims: Like most European countries, Norway has refrained from mandatory food fortification with folic acid to reduce the number of neural tube defects. We explored the role of folic acid and multivitamin supplements in the prevention of neural tube defects among newborn infants. Methods: We used data from the Medical Birth Registry of Norway, 1999–2013. A total of 528,220 women had 880,568 pregnancies resulting in 896,674 live- and stillborn infants, of whom 270 had neural tube defects. Relative risks were estimated with log-binomial regression. Results: From 1999 to 2013, intake of folic acid supplements increased from 4.8% to 27.4%. Vitamin supplement use was more frequent in older, married or cohabiting women and those with lower parity, as well as women who did not smoke during pregnancy. The overall adjusted relative risk of infant neural tube defects associated with maternal vitamin intake before pregnancy relative to no intake was 0.76 (95% confidence interval: 0.53–1.10). When we divided our study period in two (1999–2005 and 2006–2013), we found a significantly reduced risk of neural tube defects overall by vitamin use in the second time period, but not in the first: adjusted relative risk 0.54 (95% confidence interval: 0.31–0.91) and 1.02 (95% confidence interval: 0.63–1.65), respectively. Conclusions: Over the full study period, we found no statistically significant association between vitamin use and neural tube defects overall. However, vitamin use was associated with a significantly lower risk of neural tube defects in the second half of the study period, 2006–2013.