Objective— High-mobility group box 1 (HMGB1), a DNA-binding cytokine expressed mainly by macrophages, contributes to lesion progression and chronic inflammation within atherosclerotic plaque. It has been suggested that different cytokines could regulate HMGB1 expression in monocytes. We have analyzed the effect of tumor necrosis factor–like weak inducer of apoptosis (TWEAK) on HMGB1 expression both in vivo and in vitro. Methods and Results— Expression of TWEAK and its receptor fibroblast growth factor–inducible 14 (Fn14) was positively correlated with HMGB1 in human carotid atherosclerotic plaques. TWEAK increased HMGB1 mRNA expression and protein secretion in human acute monocytic leukemia cell line cultured monocytes. TWEAK-mediated HMGB1 increase was only observed in M1 macrophages but not in M2 ones. These effects were reversed using blocking anti-Fn14 antibody or nuclear factor-kappa B and phosphotidylinositol-3 kinase inhibitors. TWEAK also increased monocyte chemoattractant protein-1 secretion in human acute monocytic leukemia cell line cells, an effect blocked with an HMGB1 small interfering RNA. Systemic TWEAK injection in ApoE −/− mice increased HMGB1 protein expression in the aortic root and mRNA expression in total aorta of ApoE −/− mice. Conversely, TWEAK-blocking antibodies diminished HMGB1 protein and mRNA expression compared with IgG-treated mice. Conclusion— Our results indicate that TWEAK can regulate expression and secretion of HMGB1 in monocytes/macrophages, participating in the inflammatory response associated with atherosclerotic plaque development.