Objective: Increases in extracellular [Ca2+] elicit an endothelium-dependent vasodilatation through activation of the extracellular Ca2+- sensing receptor (CaSR). The CaSR is a calpain substrate and activation of the protease e.g. in diabetes mellitus is associated with the truncation of the CaSR. Since modulation of the extracellular [Ca2+] influences multiple pathways, we studied the vasodilatory properties of NPS R-568, a calcimimetic agent that enhances the sensitivity of the CaSR to Ca2+, in healthy and pre-diabetic animals. Methods: Segments of thoracic aortae from healthy mice and mice fed a western-type diet (28 weeks) were precontracted with phenylephrine and vasodilatation in response to Ca2+, NPS R-568, its less active enantiomer S- 568, as well as to acetylcholine (ACh) were studied. Calpain activation in the aorta was assessed by Western blotting. Results: In normal healthy aortae, step-wise increments in extracellular [Ca2+] from 0.5 to 5 mM elicited up to 58% vasodilatation that was prevented by the CaSR antagonist NPS 2390, L-NAME, or removal of the endothelium. NPS R-568 likewise induced a concentration- and Ca2+-dependent vasodilatation (10 mM induced 32% relaxation in the presence of 1.0 mM Ca2+ and 55% in the presence of 1.6 mM Ca2+). In contrast, S-568 did not elicit a noticeable response. Mice fed a Western-type diet developed a ~40% higher body mass index (P<0.001) but only moderate changes in glucose tolerance. Overall endothelial function, expressed as relaxation to ACh, was also unchanged. In contrast, R-568-induced dilatation was markedly attenuated in these mice (47% versus 77% in healthy mice, P = 0.046). Furthermore, in mice fed a western-type diet, m-calpain in the aorta was activated. Conclusion: Activation of the CaSR elicits the relaxation of mouse aorta but this effect is blunted in diabetic animals as a consequence of the limited proteolysis of the CaSR by calpain.