The osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) cytokine system, not only controls bone homeostasis, but has been implicated in regulating vascular calcification. TNF-related apoptosis-inducing ligand (TRAIL) is a second ligand for OPG, and although its effect in vascular calcification in vitro is controversial, its role in vivo is not yet established. This study aimed to investigate the role of TRAIL in vascular calcification in vitro using vascular smooth muscle cells (VSMCs) isolated from TRAIL(-/-) and wild-type mice, as well as in vivo, in advanced atherosclerotic lesions of TRAIL(-/-)ApoE(-/-) mice. The involvement of OPG and RANKL in this process was also examined. TRAIL dose-dependently inhibited calcium-induced calcification of human VSMCs, while TRAIL(-/-) VSMCs demonstrated accelerated calcification induced by multiple concentrations of calcium compared to wild-type cells. Consistent with this, RANKL mRNA was significantly elevated with 24 h calcium treatment, while OPG and TRAIL expression in human VSMCs was inhibited. Brachiocephalic arteries from TRAIL(-/-)ApoE(-/-) and ApoE(-/-) mice fed a high fat diet for 12 w demonstrated increased chondrocyte-like cells in atherosclerotic plaque, as well as increased aortic collagen II mRNA expression in TRAIL(-/-)ApoE(-/-) mice, with significant increases in calcification observed at 20 w. TRAIL(-/-)ApoE(-/-) aortas also had significantly elevated RANKL, BMP-2, IL-1β, and PPAR-γ expression at 12 w. Our data provides the first evidence that TRAIL deficiency results in accelerated cartilaginous metaplasia and calcification in atherosclerosis, and that TRAIL plays an important role in the regulation of RANKL and inflammatory markers mediating bone turn over in the vasculature.