Vascular calcification is a pathological process of deposition of calcium and phosphate crystals in the form of hydroxyapatite (HAp) in the blood vessel wall. Its presence in the vessel wall causes stiffness and leads to left ventricular hypertrophy and increases cardiovascular morbidity and mortality. It is a serious health problem common in ageing populations and widely prevalent in pathological conditions such as diabetes and chronic kidney disease. Vascular calcification is a regulated, cell-mediated process, similar to bone formation that involves osteogenic transdifferention of vascular smooth muscle cells (VSMCs) characterized by expression of bone-specific genes in the calcified vessel. However, the exact mechanisms responsible for triggering this process are unknown. The endoplasmic reticulum (ER) is involved in the production, correct folding and secretion of newly synthesized proteins in cells. ER stress occurs as a result of unfolded protein accumulation or an increased demand for protein secretion. It results in activation of a signalling pathway called the unfolded protein response (UPR), mediated by three main ER stress transducers IRE1, PERK and ATF6. Importantly, ER stress has recently been implicated in bone development. Therefore, we hypothesized that phenotypic conversion and calcification of VSMCs can be regulated by ER stress. Human primary VSMCs were treated with tunicamycin and thapsigargin and expression of bone markers in response to ER stress was examined by Western blotting and quantitative real-time PCR. ER stress was shown to regulate expression of BMP2, a key bone morphogen and Osterix, an obligate bone specific transcription factor, as well as its downstream targets bone sialoprotein, alkaline phosphatase and osteoprotegerin in VSMCs. However no effects on Runx2 were observed. In addition, the two different ER stress inducers were found to have differential effects on activation of these bone markers. Using siRNA knock-down it was established that two branches of the UPR represented by ATF6 and ATF4 (downstream of PERK) play a role in mediating these effects on bone gene expression. Further analysis of Osterix, the osteogenic transcription factor shown to be regulated by ER stress, demonstrated that it localised predominantly in cytoplasmic foci and was present in matrix vesicles secreted by VSMCs suggesting it may play a novel role in mediating the VSMC response to ER stress.