To assess the serum folate and vitamin B12 status in healthy Iranian adults, we designed a population-based cross-sectional study of 1200 individuals aged 20-80 years. Finally 984 participants (507 men and 477 women) were assessed. The mean serum folate was 4.61 (SD 2.40) ng/mL and the mean serum vitamin B12 level was 265.6 (SD 170.9) pg/mL. Overall 1.0% were folate deficient and 25.8% had low vitamin B12 levels according to the manufacturer's reference ranges (folate < 1.5 ng/mL and vitamin B12 < 160 pg/mL). The mean serum folate and vitamin B12 levels were significantly lower in men. The prevalence of vitamin B12 deficiency was considerably higher than folate deficiency. Implementation of preventive measures seems to be necessary.