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INTRODUCTION: The International Society of Pediatric and Adolescent Diabetes (ISPAD) suggests the initiation of metformin for the management of conditions characterized by insulin resistance and normalization of menstrual disturbances in adolescent girls with Polycystic Ovary Syndrome. The adverse effect of the aforementioned treatment is the manifestation of biochemical vitamin B12 deficiency, which emerged mainly in studies conducted on adults. OBJECTIVE: The purpose of this case- control study was to evaluate the vitamin B12 status in pediatric metformin-receiving patients, compared to their peers, who were not administered this medical substance. RESULTS: Children on metformin were older (p<0,001), and had higher weight (p≤ 0,001) and height (p≤ 0,001) than those who were not on treatment. However, no difference was noted in their BMI (z-scores). Concentrations of Vitamin B12, 25(OH)Vitamin D, Fasting Glucose and Insulin, Calcium, SHBG and HOMA-IR did not differ between patients receiving or not metformin. However, participants in the first group had lower concentrations of Phosphorus (p≤0,001) and Alkaline Phosphatase (p<0,001) and higher levels of d4 Androstenedione (p<0,001) and DHEA-S (p<0,001) than those who were not receiving treatment. Biochemical Vitamin B12 deficiency (<140 pg/ml) was detected only in 4 members (17%) of the patients group. In this group, a higher dietary intake of Vitamin B12 was recorded, which came entirely from dietary sources, since none of its members took any cobalamin-enriched oral nutritional supplement. This finding probably compensated for the low serum B12 levels, which may have occurred due to the administered treatment. CONCLUSIONS: Despite the absence of a statistical significant difference in cobalamin concentrations between patients and controls and the higher Vitamin B12 consumption by the former, biochemical deficiency was detected only in participants receiving metformin. That’s why regular monitoring of Vitamin B12 levels is recommended in pediatric patients treated with metformin.