Chronic inflammation in arterial wall that is driven by immune cells and cytokines plays pivotal roles in the development of atherosclerosis. Interleukin 27 (IL-27) is a member of the IL-12 family of cytokines that consists of IL-27p28 and Epstein-Barr virus induced gene 3 (EBI3) and has anti-inflammatory properties that regulate T cell polarization and cytokine production. IL-27-deficient (Ldlr-/-Ebi3-/-) and IL-27 receptor-deficient (Ldlr-/-WSX-1-/-) Ldlr-/- mice were generated and fed with a high-cholesterol diet to induce atherosclerosis. Roles of bone marrow-derived cells in vivo and macrophages in vitro were studied using bone marrow reconstitution by transplantation and cultured peritoneal macrophages, respectively. We demonstrate that mice lacking IL-27 or IL-27 receptor are more susceptible to atherosclerosis compared with wild type due to enhanced accumulation and activation of macrophages in arterial walls. The number of circulating proinflammatory Ly6C(hi) monocytes showed no significant difference between wild-type mice and mice lacking IL-27 or IL-27 receptor. Administration of IL-27 suppressed the development of atherosclerosis in vivo and macrophage activation in vitro that was indicated by increased uptake of modified low-density lipoprotein and augmented production of proinflammatory cytokines. These findings define a novel inhibitory role for IL-27 in atherosclerosis that regulates macrophage activation in mice.