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Nutritional intervention is effective in reducing the risk of neural tube defects (NTDs). To determine the effects of nutritional supplementation on human metabolism, a metabonomic study was carried out on 96 women of reproductive age. Subjects with nutritional intervention were given fortified wheat flour (containing folic acid, vitamin B₁, vitamin B₂, ferric sodium edetate and zinc oxide) for 8 months. Serum metabolic fingerprinting was detected via ultraperformance liquid chromatography in tandem with time-of-flight mass spectrometry (UPLC-TOF MS), and data acquired was processed by multivariate statistical analysis. The result revealed a significant difference between the control and intervention group. Twenty potential biomarkers, including fructose 6-phosphate, sphingosine 1-phosphate, docosahexaenoic acid and hexadecanoic acid, were located and identified by the accurate mass measurement of TOF MS. These compounds are believed to be functionally related to anti-oxidative competence in vivo. In conclusion, metabonomics study is a valuable approach in exploring the effect mechanism of nutritional intervention on NTD prevention., (Copyright © 2010 John Wiley & Sons, Ltd.)