Irisin, a recently identified myokine, is involved in the protection of mice against obesity and diabetes. This study aims to determine the serum and vitreous concentrations of irisin in patients with diabetic nephropathy and diabetic retinopathy. A total of 178 patients with type 2 diabetes mellitus, as well as 22 type 2 diabetes mellitus patients without diabetic retinopathy and 35 patients with proliferative diabetic retinopathy were enrolled in this study. Serum irisin concentrations were significantly elevated in the control group compared with those in type 2 diabetes mellitus patients. Furthermore, type 2 diabetes mellitus patients with macroalbuminuria exhibited significantly lower serum irisin concentrations than the controls and type 2 diabetes mellitus patients with normoalbuminuria and microalbuminuria. Simple regression analysis showed that the serum irisin concentrations in type 2 diabetes mellitus patients were negatively correlated with age, fasting plasma glucose, homeostasis model assessment of insulin resistance, blood urea nitrogen, creatinine, and positively correlated with creatinine clearance and angiotensin-converting enzyme inhibitors/angiotensin receptor blockers treatment. Proliferative diabetic retinopathy patients showed significantly decreased serum and vitreous irisin concentrations compared with the control group and type 2 diabetes mellitus patients without diabetic retinopathy. Furthermore, decreased serum and vitreous irisin concentrations were found in type 2 diabetes mellitus patients without diabetic retinopathy than those in the controls. Irisin concentrations are associated with the presence of diabetic nephropathy and diabetic retinopathy.