Many studies indicate a crucial role of vitamin B12 in hyperhomocysteinemia and hyperglicemia. In this study, through dietary restriction, we developed rat models of vitamin B12 deficiencies to elucidate the impact on hyperhomocysteinemia and hyperglycemia. We investigated whether there is a correlation between plasma Vitamin B12 with homocysteine and the presence of hyperglycemia in male rats. Adult male mice were divided into 3 groups (a) Control diet for 16 weeks (b) vitamin B12-restricted diet with pectin as dietary fiber for 8 weeks (K1) (c) vitamin B12-restricted diet with pectin as dietary fiber for 16 weeks (K2). After 8 weeks of feeding, plasma concentrations of vitamin B12, homocysteine, and glucose were assessed. Blood samples for vitamin B12, homocysteine and glucose were obtained after overnight fasting. After 16 weeks of continued feeding on their respective diets, plasma concentrations of vitamin B12, homocysteine, and glucose were assessed. Plasma vitamin B12 levels were lower in K2 compared to both the control and K1 groups. On the other hand, the deficient B12 group had significantly higher levels of homocysteine and glucose than control on 8 and 16 weeks. In this study, we concluded that hyperglycemia was associated with lower serum vitamin B12 concentration and serum homocysteine in the development of vitamin B12 deficient rat model.