Calcium deposits are commonly found in association with ageing and with several diseases including atherosclerosis, medial calcification (Mönckeberg’s sclerosis), arthritis and cancer. In bone, nucleation of calcium crystals occurs in matrix vesicles which are nanoparticulate, membrane-bound structures released from osteoblasts and chondrocytes. Similar structures released from vascular smooth muscle cells (VSMCs) have been detected in human calcified arteries and from VSMCs in vitro. The amount of calcium phosphate (CaP) crystals deposited in arteries correlates positively with atherosclerotic plaque rupture and myocardial infarction but whether the crystals actively participate in driving the disease is unclear. The damaging effect of nano- and microparticulate CaP crystals in arthritic joints has been known for some time and recent studies in human atherosclerotic plaques suggest that very small CaP crystals occur in regions of stress and plaque rupture. VSMCs have a role in protecting atherosclerotic plaques from rupture by forming a thick fibrous cap. To investigate whether CaP crystals could affect VSMC function, we added either synthetic crystals or calcified particles extracted from human atherosclerotic plaques to human aortic VSMCs in culture. Both types of crystals induced cell death in VSMCs, with the synthetic crystals being more potent than the plaque-extracted crystals. To investigate the mechanism of cell death, intracellular calcium ion levels were measured using video imaging of Fura-2-loaded cells. CaP crystals caused rapid rises in intracellular calcium ion concentration preceding cell death and these effects were inhibited when lysosomal acidification was blocked with bafilomycin A. This suggested that the crystals were endocytosed by VSMCs, dissolved in lysosomes and subsequent release of free calcium ions into the cytosol resulted in calcium overload and cell death. By imaging individual cells, we have observed that although CaP crystals induce cell death in VSMCs, the response is not uniform, with some cells responding to the toxic insult rapidly, while others are more resistant. This may be due to the heterogeneous nature of VSMCs. To investigate why plaque-extracted crystals are less potent than synthetic crystals, we tested the hypothesis that calcification-associated proteins such as fetuin could influence crystal toxicity. We found that fetuin inhibited rises in intracellular calcium and cell death induced by the CaP crystals, in a dose-dependent manner (in the range 0.1-10mM). Uncovering how CaP crystals can impact on VSMC survival and function may provide strategies to limit their damaging effects in the vessel wall.