We evaluated the relationship of an alanine or valine polymorphism at amino acid sequence 16 [Val(16)Ala] of manganese superoxide dismutase (Mn-SOD) with diabetes and diabetic nephropathy in Japanese type 2 diabetic patients. Val(16)Ala genotyping of Mn-SOD was done by polymerase chain reaction-restriction fragment length polymorphism with a restriction enzyme (Bsaw I) in 478 Japanese type 2 diabetic patients and 261 nondiabetic Japanese healthy subjects. The genotype distribution of diabetic and nondiabetic subjects was then compared, and the association of genotype with diabetic nephropathy was evaluated in the diabetic patients. The allele frequency and genotype of the diabetic patients were not different from those of the healthy nondiabetic subjects. The VV type showed a significantly higher frequency in the diabetic patients with nephropathy than did the AA or VA type [VV type: normoalbuminuria 70.8%, microalbuminuria 84.8% (P = 0.0057), macroalbuminuria 84.1% (P = 0.0128)]. Furthermore, logistic regression analysis showed that this polymorphism is associated with diabetic nephropathy independently (odds ratio = 0.461925, P = 0.03). Accordingly, the Val(16)Ala polymorphism of Mn-SOD may be unrelated to the etiology of type 2 diabetes, but it seems to be associated with diabetic nephropathy in Japanese type 2 diabetic patients.