Metformin, an insulin-sensitizing drug, is a first line treatment for type 2 diabetes. Long-term use of metformin has been associated with subsequent reductions in vitamin B12 concentrations. The objective of our study was to determine whether metformin use is associated with lower serum vitamin B12 concentrations in older adults, and whether concurrent use of multivitamins modifies this association. We examined 2,510 participants aged 50 years and over, participating in the national population-based Reasons for Geographic And Racial Differences in Stroke (REGARDS) Study. Multivariable linear and logistic regression models were used to assess associations between multivitamin use and serum vitamin B12 concentrations. We estimated adjusted odds ratios (aOR)s and confidence intervals (CI)s. Results were stratified by three metformin/diabetes sub-groups: 1) participants with diabetes who were metformin users; 2) participants with diabetes who were not metformin users; and 3) participants without diabetes. We found that diabetic metformin users had significantly lower geometric mean serum B12 concentrations (409 pmol/L) than the group with diabetes not taking metformin (485 pmol/L; P<0.01), and the group without diabetes (445 pmol/L; P = 0.02). The geometric mean serum B12 concentrations were greater for multivitamin users (509 pmol/L) compared to those who did not use multivitamins (376 pmol/L; p<0.01). Among the participants with diabetes who were on metformin therapy, multivitamin use was associated with geometric mean serum vitamin B12 concentrations that were 50% (or 161 pmol/L) higher, compared to those not using multivitamins. Among metformin users, multivitamin use was associated with lower prevalence of combined low and borderline vitamin B12 concentrations (aOR = 0.14; 95% CI = 0.04, 0.54) compared to those not using multivitamins. In conclusion, metformin use was associated with lower geometric mean serum vitamin B12 concentrations among diabetic older adults compared to their counterparts. Concurrent multivitamin use may potentially protect against low or borderline vitamin B12 concentrations in long-term metformin users. Additional research is needed to further examine this association as low or borderline vitamin B12 concentrations can be preventable, or treatable if detected at an early stage, in long-term metformin users.