Vasostatins (VSs) are vasoactive peptides derived from chromogranin A (CgA), a protein contained in secretory granules of chromaffin and other cells. The negative inotropic effect and the reduction of isoproterenol-dependent inotropism induced by VSs in the heart, suggest that they have an anti-adrenergic function. However, the mechanisms of action of VSs need further investigation. Our study aims at defining the signaling pathways activated by VS-1 in mammalian ventricular myocardium and in cultured endothelial cells that lead to the modulation of cardiac contractility. The effects induced by recombinant human VS-1 (STA-CgA1-76) on contractile force, calcium current (I_Ca,L) and calcium transients were studied under basal conditions and upon beta-adrenergic stimulation in rat papillary muscles and ventricular cells, while those induced on intracellular calcium concentration and nitric oxide (NO) production were studied in cultured bovine aortic endothelial cells (BAE-1) using Ca²⁺ and NO fluorimetric confocal imaging. On papillary muscle VS-I was ineffective on the basal contractility, while the effect of Iso stimulation was reduced by 27%. Removal of endocardial endothelium and inhibition of NO synthesis and PI3K activity abolished the anti-adrenergic effect of VS-1 on papillary muscle. In cardiomyocytes, VS-1 (10 nM) was ineffective on basal and Iso-stimulated (1 µM) I_Ca,L and calcium transients. In BAE-1 cells, VS-1 induced a calcium independent increase in NO production, blocked by the PI3K inhibitor Wortmannin. Our results suggest that the anti-adrenergic effect exerted by VS-1 is mainly due to a PI3K dependent-NO release by endothelial cells, rather than to a direct action on cardiomyocytes.