Objective: Although an increasing number of reports suggest that physiological concentrations of C-peptide protect against the development of diabetic nephropathy, possibly through the modulation of Na–K pump activity, the intracellular pathways controlled by C-peptide are still unrecognized. C-peptide and vasopressin share similar intracellular effects including the activation of calcium influx and endothelial nitric oxide synthase. Both hormones stimulate also the activity of Na–K pump activity. Whether the activity of C-peptide is mediated by the recently identified vasopressin-activated calcium-mobilizing receptor (VACM-1) has never been previously investigated.Design and methods: To clarify this issue, we evaluated the effect of C-peptide on VACM-1 RNA (measured by semiquantitative RT-PCR) and protein expression (measured by immunoblotting) in human skin fibroblasts (where a specific binding of C-peptide was demonstrated) and in human mesangial cells, the cellular target of diabetic nephropathy.Results: C-peptide-induced activation of VACM-1 was demonstrated in fibroblasts from six healthy individuals (0.51±0.1 vs 1.48±0.4, arbitrary units±s.e.,P= 0.025). This finding was paralleled by an increased VACM-1 protein expression (5.64±1.0 vs 8.47±1.2, arbitrary units±s.e.,P= 0.043). Similar results were confirmed in three independent cultures of human mesangial cells. VACM-1 activation in fibroblasts was insensitive to phosphatidylinositol-3-kinase inhibitor LY294002, but was inhibited by pertussis toxin, suggesting that activation of VACM-1 could be mediated by a G protein-coupled receptor.Conclusions: This study demonstrates for the first time that C-peptide activates VACM-1, possibly through a G protein-coupled receptor. Further studies are needed to clarify whether VACM-1 is involved in the protective effect of C-peptide against the development of diabetic nephropathy.