OBJECTIVE: To evaluate the clinical outcomes at age 1.5 ± 0.5 years of infants with vitamin B deficiency identified by newborn screening (NBS). STUDY DESIGN: Prospective multicenter observational study on health outcomes of 31 infants with vitamin B deficiency identified by NBS. Neurodevelopment was assessed by the Denver Developmental Screening Test. RESULTS: In 285 862 newborns screened between 2016 and 2019, the estimated birth prevalence of vitamin B deficiency was 26 in 100 000 newborns, with high seasonal variations (lowest in summer: 8 in 100 000). Infants participating in the outcome study (N = 31) were supplemented with vitamin B for a median (range) of 5.9 (1.1-16.2) months. All achieved age-appropriate test results in Denver Developmental Screening Test at age 15 (11-23) months and did not present with symptoms characteristic for vitamin B deficiency. Most (81%, n = 25) mothers of affected newborns had a hitherto undiagnosed (functional) vitamin B deficiency, and, subsequently, received specific therapy. CONCLUSIONS: Neonatal vitamin B deficiency can be screened by NBS, preventing the manifestation of irreversible neurologic symptoms and the recurrence of vitamin B deficiency in future pregnancies through adequate treatment of affected newborns and their mothers. The high frequency of mothers with migrant background having a newborn with vitamin B deficiency highlights the need for improved prenatal care.