Background <p>Metformin treatment has a risk factor of reduced serum concentrations of vitamin B12 and zinc, indicating its association with homocysteine metabolism. However, this association remains to be clarified in patients with type 2 diabetes (T2DM) accompanied by kidney dysfunction.</p> Methods <p>This cross-sectional study was conducted in 149 patients with T2DM (96 men, 53 women), including diabetic kidney disease. Serum concentrations of homocysteine, as well as vitamin B12, folic acid, and zinc, were measured in outpatient T2DM patients. The study subjects were divided into two groups: patients with and without metformin administration (Met [ +], n = 62; Met [ −], n = 87). To explore the effect of kidney function, we also analyzed the data after dividing all the patients according to kidney function (chronic kidney disease [CKD] group, n = 66; non-CKD group, n = 83).</p> Results <p>The Met ( +) group exhibited significantly higher serum zinc levels and lower serum homocysteine levels than the Met ( −) group. In the non-CKD group, metformin administration was positively associated with serum zinc levels, as demonstrated by multiple linear regression analysis adjusted for confounding factors (<i>β</i> = 0.287, <i>p</i> = 0.021). However, no significant association between metformin administration and serum zinc levels was observed in the CKD group. Moreover, there were no associations between serum homocysteine levels and metformin administration.</p> Conclusions <p>The relationship between metformin treatment and serum zinc levels differed based on the presence or absence of CKD in patients with T2DM.</p>