Glucose intolerance and dyslipidaemia are independent risk factors for endothelium dysfunction and CVD. The aim of the present study was to analyse the preventive effect of n-3 PUFA (EPA and DHA) on lipid and carbohydrate disturbances and endothelial dysfunction. Three groups of adult hamsters were studied for 20 weeks: (1) control diet (Control); (2) high-fat diet (HF); (3) high-fat diet enriched with n-3 PUFA (HFn-3) groups. The increase in body weight and fat mass in the HF compared to the Control group (P < 0.05) was not found in the HFn-3 group. Muscle TAG content was similar in the Control and HF groups, but significantly lower in the HFn-3 group (P = 0.008). Glucose tolerance was impaired in the HF compared to the Control group, but this impairment was prevented by n-3 PUFA in the HFn-3 group (P < 0.001). Plasma TAG and cholesterol were higher in the HF group compared to the Control group (P < 0.001), but lower in the HFn-3 group compared to the HF group (P < 0.001). HDL-cholesterol was lower in the HFn-3 group compared to the Control and HF groups (P < 0.0005). Hepatic secretion of TAG was lower in the HFn-3 group compared to the HF group (P < 0.005), but did not differ from the Control group. Hepatic gene expression of sterol regulatory element-binding protein-1c, diacylglycerol O-acyltransferase 2 and stearyl CoA desaturase 1 was lower in the HFn-3 group, whereas carnitine palmitoyl transferase 1 and scavenger receptor class B type 1 expression was higher (P < 0.05). In adipocytes and adipose macrophages, PPARγ and TNFα expression was higher in the HF and HFn-3 groups compared to the Control group. Endothelium relaxation was higher in the HFn-3 (P < 0.001) than in the HF and Control groups, and was correlated with glucose intolerance (P = 0.03) and cholesterol (P = 0.0003). In conclusion, n-3 PUFA prevent some metabolic disturbances induced by high-fat diet and improve endothelial function in hamsters.