Background. Thiosulfate has been shown to inhibit vascular calcification in uremic rats and may inhibit calcification in humans with end-stage renal disease but whether this is due to a systemic or local action is unknown. The underlying mechanism is also unclear but complexation of calcium ions has been proposed. Methods. In vitro assays were used to determine the effect of thiosulfate on vascular calcification and hydroxyapatite formation. Results. Thiosulfate (EC50: 1–2 mM) prevented calcification of injured or devitalized aortas but not uninjured aortas, and similar results were obtained with sulfate. There was no effect on reversal of calcification. Measurements with an ion-sensitive electrode (corrected for changes in ionic strength) revealed a very weak interaction between thiosulfate and Ca2+ (Ka = 10.9 ± 1.0 × 10−6 M−1) that resulted in a 4% decrease in ionized Ca2+ in culture medium at 5 mM thiosulfate and a corresponding 5% increase in the solubility product for calcium–phosphate. Adjustment of the total Ca2+ concentration to account for this did not prevent the inhibition of aortic calcification by thiosulfate. Thiosulfate did not inhibit hydroxyapatite formation from seed crystals or the calcification of purified elastin and did not alter medium pH. Conclusions. Thiosulfate inhibits vascular calcification at millimolar concentrations through a direct extracellular effect that does not require intact smooth muscle cells but is related to cellular injury. This effect is not specific for thiosulfate since sulfate has similar properties. Inhibition cannot be explained by effects on ionized calcium, calcium–phosphate solubility, pH, oxidative stress or hydroxyapatite formation.