ArtículoAbstract Background Patients with type 2 diabetes mellitus (T2DM) are characterized by chronic hyperglycemia as a consequence of decreased insulin sensitivity, which contributes to bone demineralization and could also be related to changes in serum levels of osteocalcin and insulin, particularly when coupled with a deficiency in the daily consumption of vitamins D3 and K2. The objective of this study was to evaluate the effect of vitamin D3 and vitamin K2 supplements alone or in combination on osteocalcin levels and metabolic parameters in patients with T2DM. Methods A double-blind, randomized clinical trial was carried out in 40 patients aged between 30 and 70 years old for 3 months. Clinical and laboratory assessment was carried out at the beginning and at the end of the treatment. The patients were divided into three groups: (a) 1000 IU vitamin D3 + a calcinated magnesium placebo (n = 16), (b) 100 µg of Vitamin K2 + a calcinated magnesium placebo (n = 12), and (c) 1000 IU vitamin D3 + 100 µg vitamin K2 (n = 12). Results After treatment in the total studied population, a significant decrease in glycemia (p = 0.001), HOMA-IR (Homeostatic model assessment-insulin resistance) (p = 0.040), percentage of pancreatic beta cells (p < 0.001), uOC/cOC index and diastolic blood pressure (p = 0.030) were observed; in vitamin D3 group, differences in serum undercarboxylated osteocalcin (p = 0.026), undercarboxylated to carboxylated osteocalcin index (uOC/cOC) (p = 0.039) glucose (p < 0.001) and % of functional pancreatic beta cells (p < 0.001) were demonstrated. In vitamin K2 group a significant decrease in glycemia (p = 0.002), HOMA-IR (p = 0.041), percentage of pancreatic beta cells (p = 0.002), and in cOC (p = 0.041) were observed, conversely cOC concentration was found high. Finally, in the vitamins D3 + K2 a significant decrease in glycemia (p = 0.002), percentage of pancreatic beta cells (p = 0.004), and in the uOC/cOC index (p = 0.023) were observed. Conclusion Individual or combined supplementation with vitamins D3 and K2 significantly decreases the glucose levels and % of functional pancreatic beta cells, while D3 and D3 + K2 treatments also induced a reduction in the uOC/cOC index. Only in the group with vitamin D3 supplementation, it was observed a reduction in undercarboxylated osteocalcin while vitamin K2 increased the carboxylated osteocalcin levels.