Aims: To examine the association of homozygosity for the MTHFR C677T mutation, and vitamin B12 deficiency in 360 asymptomatic individuals and to investigate forearm endothelial function in C677T homozygotes. Methods and Results: MTHFR C677T mutation, levels of vitamin B12, folic acid and homocysteine were measured in study participants. Frequency of homozygosity for the C677T mutation was 67/360 (18.6%). Homocysteine levels were elevated in homozygous compared to heterozygous subjects or those without the mutation (20.6±18.8 vs. 9.4±3.2 µmol/L, p<0.0001). The number of subjects with vitamin B12 deficiency (<150pmol/L) was significantly higher among the homozygotes compared to heterozygotes or subjects without mutation (20/67 (29.8%) vs. 27/293, (9.2%); p<0.0001). Homozygotes had 4.2 times higher probability of having B12 deficiency (95% CI 2.1-8.3). Forearm endothelial function was assessed in 33 homozygotes and 12 controls. Abnormal endothelial function was observed in homozygous subjects and was worse in homozygotes with vitamin B12 deficiency. Endothelial function was normalized after B12 and folic acid treatment. Conclusion: Homozygosity for the C677T mutation is strongly associated with B12 deficiency. Co-existence of homozygosity for the C677T mutation and B12 deficiency is associated with endothelial dysfunction and can be corrected with vitamin B12 and folic acid treatment.