Background: Vitamin B12 deficiency is associated with hematological, neurological, and cardiovascular consequences. Epidemiologic data on these related illnesses indicate gender differences. Methods: A cross-sectional study was designed to examine gender differences in vitamin B12 deficiency among a healthy population. Data from healthy individuals aged 18–65, who were provided with a routine medical evaluation during 2000–2014, were retrieved from the medical charts. Individuals with background illnesses and those who had used medications or nutritional supplements were excluded. Vitamin B12 deficiency was defined by 2 cutoff values (206 and 140 pmol/L). The multivariate analysis was adjusted for age, body mass index, estimated glomerular filtration rate, hyperhomocysteinemia, folate deficiency, albumin, and transferrin saturation. Sensitivity analyses were implemented by excluding individuals with anemia, hyperhomocysteinemia, or folate deficiency and by age stratification. Results: In all, 7,963 individuals met the inclusion criteria. Serum vitamin B12 mean levels were 312.36 and 284.31 pmol/L for women and men respectively (p < 0.001). Deficiency prevalence was greater for men (25.5%) in comparison with women (18.9%; p < 0.001). Men were strongly associated with severe deficiency (adjusted OR 2.26; 95% CI 1.43–3.56). Conclusions: Among the healthy population, men are susceptible to vitamin B12 deficiency. This can be explained by neither diet habits nor estrogen effects. Genetic variations are therefore hypothesized to play a role.