Abstract Introduction Considering the low level of vitamin B12 in diabetic patients on Metformin and also the importance of this vitamin in lipid metabolism , the aim of the present study was assessing the possible relationship between vitamin B 12 deficiency, lipid profile and atherogenic indices in Iranian diabetic patients. Method In the present cross-sectional study, a sample of 200 patients with type 2 diabetes was selected. Serum level of lipid profile was measured using enzymatic colorimetric method. LDL-C was calculated by Friedewald equation and atherogenic indices including total cholesterol to HDL-C and Log Triglyceride to HDL-C ratio were calculated. Serum level of vitamin B 12 was measured photometrically using commercial kit. Results The prevalence of vitamin B12 deficiency was about 14%. There was a significant difference between normal participants and vitamin B12 deficient subjects in the case of total cholesterol (P = 0.04). The results of the regression analysis showed that in adjusted model, there was significant association between total cholesterol and vitamin B12 deficiency (OR: 16.46 [95% CI: 0.27, 33.19], P = 0.04). Conclusion The results of the present study showed the relatively high prevalence of the vitamin B12 deficiency in type 2 diabetic patients who were on Metformin. Moreover, there was a significant association between vitamin B12 deficiency and total cholesterol level in diabetic patients. Further studies with larger sample size and considering dietary information and duration of Metformin consumption are needed to confirm these preliminary results.