Introduction: The efficacy of the intramuscular administration of vitamin B12 is well established, but it is limited by the fact that it should be administered by a healthcare professional and the associated local injection site reaction. Aim: To investigate the effectiveness of sublingual vitamin B12 administration in patients with low serum B12 levels. Patients and Methods: This is a retrospective study including patients with low serum levels of B12 (<200 pg/mL) and treated with 1000 μg/day sublingually (B12-SOLGAR®) for 3 months. Subjects’ main characteristics and personal history were recorded, as well as laboratory parameters before and 3 months after therapy initiation. Results: 314 patients (men: 43%, age: 64 [54-75] years) were included in this study. Most participants were on metformin (48%) and/or a proton pump inhibitor/H2 antagonist (50%). The administration of sublingual vitamin B12 was associated with a significant increase in serum B12 levels (from 182 [152-196] to 403 [312-590] pg/mL, p<0.05). Likewise, a significant increase was also noted in the patients with very low vitamin B12 levels (<100 pg/mL, n=21) (from 85 [74-92] pg/mL to 298 [294-340] pg/mL, p<0.05). Furthermore, significant improvements in hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cells, and platelets were observed. Conclusions: The sublingual administration of vitamin B12 (1000 μg/day) for 3 months in patients with low B12 serum levels results in significant increases of B12 levels and improves hematologic parameters, and thus may be considered as an alternative to the intramuscular administration.