Objective: To explore (a) the risk factors associated with a deficiency of vitamin B12, and (b) the baseline (cutoff) serum level of vitamin B12 for a clinically-symptomatic deficiency in the Jordanian adult population. Background: Compared to the data available for developed countries, there is a marked scarcity of information on the levels and symptomology of vitamin B12 deficiency in developing countries, particularly in the Middle Eastern region. Methods: A total of 485 subjects were included in this study. Blood samples were drawn for biochemical analysis and data regarding socio-demographics, general health, anthropometric measures, and past medical, surgical, and medication history were collected. To explore the cut-off point, we compared all parameters included in a standard complete blood count as well as main symptoms reported to be associated with B12 deficiency between groups of different B12 cut-off values, consisting of those above and below 200, 175, 150 and 125 pg/ml. Results: Dietary habits, age, recurrent headaches, heart burn and peptic ulcer disease were found to be significantly associated with lower vitamin B12 levels. Surprisingly, daily smoking was associated with significantly higher B12 levels. The results revealed that none of the included potential indicators of B12 deficiency could be considered an indicative feature of deficiency. There were no significant differences neither in the symptoms nor in the CBC parameters between any of the tested study groups. Conclusion: Low dietary intake, older ages, recurrent headaches, heartburn and peptic ulcer disease, all could be considered as a risk factors of having low vitamin B12 levels within the Jordanians. Also, they tend to have lower levels of vitamin B12 levels, in comparison to countries in the West, without necessarily having deficiency symptoms. Cut-off value to diagnose functional B12 deficiency could be less than 125 pg/ml for the Jordanians. More local studies are needed to establish an accurate vitamin B12 cut-off value for the population in Jordan.