The aim of this study was to evaluate the possible association between serum fatty acids composition and endothelial dysfunction in patients with type 2 diabetes mellitus. A cross-sectional study was conducted with 125 normo- or microalbuminuric type 2 diabetes mellitus patients with serum creatinine <1.5 mg/dL. Serum fatty acids composition (gas chromatography), serum levels of endothelin-1 (ET-1) (enzyme-linked immunosorbent assay), fibrinogen, serum C-reactive protein, lipids, homeostasis model assessment resistance index (HOMA-R), and 24-hour urinary albumin excretion rate were measured. Serum levels of ET-1 were positively correlated with saturated fatty acids (r = 0.257, P = .025) and negatively correlated with polyunsaturated fatty acids (PUFAs) (r = -0.319, P = .005). Serum ET-1 levels were also positively correlated with systolic blood pressure, waist circumference, total cholesterol levels, triglycerides, and HOMA-R. In multiple linear regression models, only saturated fatty acids (R(2) = 0.317, P = .002) or PUFAs (R(2) = 0.314, P = .001) remained associated with ET-1 levels. Models were adjusted for systolic blood pressure, HOMA-R, waist circumference, triglycerides, body mass index, and smoking habit. The serum total PUFA levels showed an inverse correlation with urinary albumin excretion rate (r = -0.248, P = .012). In conclusion, in type 2 diabetes mellitus patients, the serum fatty acids composition was independently related to endothelial function evaluated by serum ET-1. Saturated fatty acids were associated with endothelial dysfunction (high levels of ET-1), whereas PUFAs had a protective role in endothelial function.