The molecular basis of atherosclerosis is associated with excessive proliferation of vascular cells. Previous studies have suggested an inverse correlation between the expression of the growth suppressor p27Kip1 (p27) and cellular proliferation within human atherosclerotic tissue. However, no causal link between diminished p27 expression and atherogenesis has been established. We investigated the effect of p27 inactivation on diet-induced atherogenesis. We find that p27-deficient mice challenged with a high-fat diet for 1 month remain normocholesterolemic and have essentially no visible atheromas. However, when generated in an apolipoprotein E-null genetic background that leads to severe hypercholesterolemia in response to the atherogenic diet, deletion of p27 enhances arterial cell proliferation (∼fourfold) and accelerates atherogenesis (∼sixfold) compared with apolipoprotein E-deficient mice with an intact p27 gene. Analysis of apolipoprotein E-null mice bearing only one p27 allele inactivated reveals that a moderate decrease in p27 protein expression in the setting of hypercholesterolemia is sufficient to predispose to atherogenesis. Thus, our study establishes a molecular link between decreased p27 protein expression and atherogenesis in hypercholesterolemic animals.—Díez-Juan, A., Andrés, V. The growth suppressor p27Kip1 protects against diet-induced atherosclerosis.