Background: Vitamin B12 deficiency is highly prevalent worldwide and has been linked to hematologic, neurologic and psychiatric diseases. There are a few studies regarding vitamin B12 deficiency in developing countries in general and in Jordan in particular. Objectives: The aims of the present study were to assess the vitamin B12 status of Jordanians at national level and to identify population groups at high risk for vitamin B12 deficiency. Methods: Vitamin B12 status was assessed in a national sample of 5,640 subjects aged >8 years. The study involved interviews, laboratory measurements of vitamin B12 and other parameters, and physical measurements. The present report deals exclusively with subjects aged >18 years (n = 2,847). Results: The percentages of subjects with low (<200 pg/ml, n = 857), borderline (201-350 pg/ml, n = 382) and normal vitamin B12 level (>350 pg/ml, n = 1,608) were 30.1, 13.4 and 56.5%, respectively. Of the 382 subjects who had borderline vitamin B12 level, 61 subjects had both increased total homocysteine (tHcy; >13 µmol/l) and low holocobalamin (<35 pmol/l). Since elevated tHcy also indicates folate deficiency, the overall prevalence of vitamin B12 deficiency reached 32.2% (31.9% among males and 32.4% among females) after adding those 61 subjects to the 857 subjects with low vitamin B12 level. Conclusion: In conclusion, our study showed that almost one third of Jordanian adults have vitamin B12 deficiency with no gender differences. Intake of vitamin B complex and multivitamins seems to protect from vitamin B12 deficiency. An in-depth study of the dietary and eating habits of Jordanians may be needed to explain the observed age and regional differences in vitamin B12 deficiency in the study population.