Chronic recruitment of monocytes and their subsequent migration through the activated endothelium contribute to atherosclerotic plaque development. Integrin-mediated leukocyte adhesion is central to this process. Conjugated linoleic acid (CLA) has the unique property of inducing regression of pre-established murine atherosclerosis via modulation of monocyte/macrophage function. Understanding the mechanisms through which CLA mediates its atheroprotective effect may help to identify novel pathways that limit or reverse atherosclerosis. In this study, we identified a novel mechanism through which CLA alters monocyte function. We show that CLA inhibits human peripheral blood monocyte cell adhesion to activated endothelial cells via loss of CD18 expression, the β2 chain of LFA-1 and Mac-1 integrins. In addition, using a static-adhesion assay, we provide evidence that CLA prevents monocytes from binding to ICAM-1 and subsequently reduces the capacity of these cells to polarize. CXCL12-CXCR4 interactions induce a conformational change in β2 integrins, facilitating leukocyte adhesion. In this study, we demonstrate that CLA inhibits CXCR4 expression, resulting in a failure of monocytes to directionally migrate toward CXCL12. Finally, using intravital microscopy, we show that, during CLA-induced regression of pre-established atherosclerosis in ApoE(-/-) mice, there is reduced leukocyte adhesion and decreased CD18 expression on Gr1(+)/CD115(+) proinflammatory monocytes. In summary, the data presented describe a novel functional role for CLA in the regulation of monocyte adhesion, polarization, and migration.