Background: The important role of vitamin D3 in human health is well recognized. In this study, we measured serum concentrations of vitamin D3, vitamin B12 and B9 (folic acid) in 410 women undergoing in vitro fertilisation (IVF)/intracytoplasmatic sperm injection (ICSI) with dedicated focus on 3-month changes in consideration of patients’ BMI. Methods: Patients were of European origin and did not take any supplementation of D3. In preparing for pregnancy, patients took ≥4 weeks 400 µg folic acid combined with 9 µg vitamin B12 and 150 µg iodide as recommended. Results: We found a significant 3-month quartile change of D3 serum concentrations (p < 0.0001) with maximum levels in autumn and lowest in spring. D3 correlated significantly with B12 (p = 0.035, ρ = 0.102) and folic acid (p < 0.0001, ρ = 0.191). BMIs however showed a negative correlation with B12 (p = 0.031, ρ = –0.105) and folic acid (p = 0.012, ρ = –0.125). Conclusions: Our results suggest a model in which the sun exposure during summer months enables storage of D3 followed by a slow release as a major factor to maintain D3 levels throughout the year. Finally, our data indicate that B12 and folic acid uptake might be influenced by vitamin D receptor and D3, where D3 and the BMI appear to have an indirect relationship – via B12 and folic acid.