Methylenetetrahydrofolate reductase (MTHFR) polymorphism has been shown to be associated with the development of diabetic nephropathy in many ethnic groups. In this study, we examined the correlation between MTHFR C677T polymorphism and microalbuminuria in patients with diabetes mellitus type 2 in Croatian patients. 85 patients with diabetes mellitus type 2 were recruited. Patients were classified into two groups--with and without diabetic nephropathy according to urinary albumin excretion rate in urine collected during 24 hours. The C677T genotype was determined by real-time PCR analysis. The genotype frequencies were CC 36.5%, CT 42.3% and TT 21.2% in diabetic patients without nephropathy versus CC 39.4%, CT 45.4% and TT 15.2% in those with nephropathy. There was no statistically significant difference in allele distribution between patients with nephropathy and those without (p = 0.788). Our study did not show a correlation between mutations in the MTHFR gene and diabetic nephropathy in Croatian patients. Diabetic nephropathy is influenced by multiple risk factors which can modify the importance of MTHFR polymorphism in its development.