Background: The biological functions of high-density lipoproteins (HDLs) contribute to explaining the cardioprotective role of the lipoprotein beyond quantitative HDL cholesterol levels. A few small-scale interventions with a single antioxidant have improved some HDL functions. However, to date, no long-term, large-scale, randomized controlled trial has been conducted to assess the effects of an antioxidant-rich dietary pattern (such as a traditional Mediterranean diet [TMD]) on HDL function in humans. Methods: This study was performed in a random subsample of volunteers from the PREDIMED Study (Prevención con Dieta Mediterránea; n=296) after a 1-year intervention. We compared the effects of 2 TMDs, one enriched with virgin olive oil (TMD-VOO; n=100) and the other enriched with nuts (TMD-Nuts; n=100), with respect to a low-fat control diet (n=96). We assessed the effects of both TMDs on the role of HDL particles on reverse cholesterol transport (cholesterol efflux capacity, HDL ability to esterify cholesterol, and cholesteryl ester transfer protein activity), HDL antioxidant properties (paraoxonase-1 arylesterase activity and total HDL antioxidant capacity on low-density lipoproteins), and HDL vasodilatory capacity (HDL ability to induce the release of nitric oxide in endothelial cells). We also studied the effects of a TMD on several HDL quality-related characteristics (HDL particle oxidation, resistance against oxidative modification, main lipid and protein composition, and size distribution). Results: Both TMDs increased cholesterol efflux capacity relative to baseline (P=0.018 and P=0.013 for TMD-VOO and TMD-Nuts, respectively). The TMD-VOO intervention decreased cholesteryl ester transfer protein activity (relative to baseline, P=0.028) and increased HDL ability to esterify cholesterol, paraoxonase-1 arylesterase activity, and HDL vasodilatory capacity (relative to control, P=0.039, P=0.012, and P=0.026, respectively). Adherence to a TMD induced these beneficial changes by improving HDL oxidative status and composition. The 3 diets increased the percentage of large HDL particles (relative to baseline, P<0.001). Conclusions: The TMD, especially when enriched with virgin olive oil, improved HDL atheroprotective functions in humans. Clinical Trial Registration: URL: http://www.controlled-trials.com. Unique identifier: ISRCTN35739639.