ADP ribosyl-cyclase (ADPR-cyclase) produces a Ca²⁺ mobilizing second messenger, cyclic ADP-ribose (cADPR), from NAD⁺. In this study, we investigated molecular basis of ADPR-cyclase activation in Angiotensin II (Ang II) signaling pathway and cellular responses in adult rat cardiomyocytes. The results showed that Ang II generated a biphasic [Ca²⁺]_i increases that include a rapid transient Ca²⁺ elevation via IP₃ receptor, and sustained Ca²⁺ rise via the activation of L-type Ca²⁺ channel and opening of ryanodine receptor (RyR). Ang II-induced sustained Ca²⁺ rise was blocked by a cADPR antagonistic analog, 8-Bromo-cADPR, indicating that sustained Ca²⁺ rise is mediated by cADPR. Supporting the notion, ADPR-cyclase activity and cADPR production by Ang II were increased in a time-dependent manner. Application of pharmacological inhibitors and immunologic analyses revealed that cADPR formation was activated by sequential activation of Src, phosphatidylinositol-3-kinase (PI3K)/Akt, phospholipase C (PLC)-1 and IP₃-mediated Ca²⁺ signal. Inhibitors of these signaling molecules not only completely abolished the Ang II-induced Ca²⁺ signals, but also inhibited cADPR formation. Application of the cADPR antagonist and inhibitors of upstream signaling molecules of ADPR-cyclase inhibited Ang II-stimulated hypertrophic responses, that include nuclear translocation, of Ca²⁺/calcineurin-dependent nuclear factor of activated T cells (NFAT)3, protein expression of transforming growth factor-1, and incorporation of [³H]leucine in cardiomyocytes. Taken together, these findings suggest that activation of ADPR-cyclase by Ang II entails a novel signaling pathway involving sequential activation of Src, PI3K/Akt, and PLC-1/IP₃, and that the activation of ADPR-cyclase can lead to cardiac hypertrophy.