Adequate vitamin B12 (B12) and folate concentrations are essential for neural development in early childhood, but studies in well-nourished children are lacking. We investigated the relation between plasma B12 and folate at 9 and 36 months and psychomotor development at 36 months in well-nourished Danish children. Subjects from the SKOT cohorts with B12 measurement and completed Ages and Stages Questionnaire, 3rd edition (ASQ-3) at 36 months were included (n 280). Dietary intake, B12 and folate concentrations were collected at 9 and 36 months, and ASQ-3 was assessed at 36 months. Associations between B12 and folate at 9 and 36 months and ASQ-3 were analysed using regression models. Associations between diet and B12 were also investigated. No children had insufficient B12 (<148 pmol/l) at 36 months. B12 at 36 month was positively associated with total ASQ-3 corresponding to an increase of 100 pmol/l B12 per 1·5 increase in total ASQ-3 score (P = 0·019) which remained significant after adjustment for potential confounders including 9 months values. B12 at 9 months or folate at any time point was not associated with total ASQ-3. Intake of milk products was associated with B12 at 36 months (P = 0·003) and showed a trend at 9 months (P = 0·069). Intake of meat products was not associated with B12. In conclusion, B12 was positively related to psychomotor development at 3 years in well-nourished children, indicating that the impact of having marginally low B12 status on psychomotor development in well-nourished children should be examined further.