Ca2+ signaling plays a fundamental role in cardiac hypertrophic remodeling, but the underlying mechanisms remain poorly understood. We investigated the role of Ca2+-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca2+ transients followed by sustained Ca2+ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca2+ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca2+ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca2+ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca2+ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively.