Increasing evidence indicates that replicative senescence and premature endothelial senescence could contribute to endothelial dysfunction and vascular disease. This study aims at testing the hypothesis that a high-fat diet may lead to premature vascular endothelial senescence in a nonhuman primate model. We isolated endothelial cells from left and right femoral arteries in 10 baboons before and after a 7-week high-fat dietary treatment. We compared the morphological alterations, replicative capacities, and senescence-associated -galactosidase activities (SA--gal) at these two time points. We found that high-fat diet increased the prevalence of endothelial senescence. Endothelial replicative capacities declined dramatically and SA--gal activities increased significantly in post-dietary challenge. There was no change in telomeric length using quantitative flow fluorescence in situ hybridization analysis, suggesting that some stressors lead to cell senescence independent of telomere dysfunction. Our findings that high-fat diet causes endothelial damage through the premature senescence suggest a novel mechanism for the diet-induced endothelial dysfunction.