We have investigated the potential role of CD1d-restricted natural killer T (NKT) cells in the development of atherosclerosis in mice. When fed an atherogenic diet (AD), NKT cell-deficient CD1d^-/- mice had significantly smaller atherosclerotic lesions than AD-fed C57BL/6 (wild-type [WT]) mice. A significant reduction in atherosclerotic lesions was also demonstrated in AD-fed, low-density lipoprotein receptor-deficient (Ldlr^-/-) mice reconstituted with CD1d^-/- bone marrow cells compared with the lesions observed in Ldlr^-/-mice reconstituted with WT marrow cells. In addition, repeated injections of α-GalCer or the related glycolipid OCH to apolipoprotein E knockout (apoE^-/-) mice during the early phase of atherosclerosis significantly enlarged the lesion areas compared with mice injected with vehicle control. However, administering α-GalCer to apoE^-/- mice with established lesions did not significantly increase the lesion area but considerably decreased the collagen content. Atherosclerosis development in either AD-fed WT or apoE^-/- mice was associated with the presence of Vα14Jα18 transcripts in the atherosclerotic arterial walls, indicating that NKT cells were recruited to these lesions. Thioglycolate-elicited macrophages pulsed with oxidized low-density lipoproteins expressed enhanced CD1d levels and induced NKT cells to produce interferon-γ, a potentially proatherogenic T-helper 1 (T_H1) cytokine. Collectively, we conclude that NKT cells are proatherogenic in mice. (Blood. 2004;104:2051-2059)