Aims: There is some clinical evidence of the association between vitamin B12 deficiency and vasovagal syncope (VVS) in pediatric patients. We aimed to investigate the possible association of vitamin B12 and folate deficiency with VVS in adults. Methods: In this case-control study, we included adult patients with VVS who presented to our tertiary syncope unit for a head-up tilt table test as the case group. We selected age- and sex-matched individuals without any history of syncope from the population-based Tehran Cohort Study as the control group. The exclusion criteria included but were not restricted to taking vitamin B supplements, carbamazepine, or phenobarbital, and sleeve gastrectomy. We measured and compared serum levels of vitamin B12, folate, and homocysteine. Results: From February 2020 to February 2021, we included 44 patients in the case group, matched with 44 controls, with a mean age of 37.9 years and 23 (52.3%) females in each group. There was no statistically significant difference between the study groups regarding vitamin B12 or folate deficiency, or their serum levels. The serum level of vitamin B12 was remarkably lower in patients with frequent VVS (≥3 lifetime episodes) compared to patients with infrequent VVS (<3 lifetime episodes) [233.8 (80.7) versus 305.2 (118.1) pg/mL; P=0.042] and this association remained significant after adjustment for possible confounders (P=0.026). Conclusion: We found no association between vitamin B12 or folate deficiency, or their serum levels and VVS; nevertheless, frequent VVS, compared to infrequent VVS, was associated with a lower serum vitamin B12.