During ischemia, the increase of [Ca2+]i activates a number of cytosolic phospholipases, proteases, and endonucleases, and then these activated proteins induce apoptotic cell death. Therefore, regulation of Ca2+ overload regulation is considered to be the key point in the recovery of myocardial hypoxic injury. We prepared hypoxic culture media (HCM) of MSCs to examine whether secreted materials from MSCs regulate [Ca2+]i in hypoxic cardiomyocytes. HCM-treated hypoxic cardiomyocytes showed 13% decrease of [Ca2+]i compared to non-treated cells. Significant induction and suppression was observed in transcription levels of Ca2+ handling proteins (calreticulin, calmodulin), ion channel (L-type Ca2+-channel), and ion exchangers (SERCA2a, Na+K+ATPase, Na+/Ca+ exchanger) in HCM-treated hypoxic cardiomyocytes. In addition, HCM-treated cells showed an increased Bcl-2/Bax ratio, and inhibition of cytochrome C signal and caspase-3 activation. Survival rate of HCM-treated hypoxic cardiomyocytes was increased about 1.6-fold and phosphorylation of ERK1/2 and Akt was increased about 3- and 2- folds, respectively. In vivo, after heart ligation, an intraperitioned injection of HCM led to 15% decrease of fibrotic heart area but also significantly reduced apoptotic positive index by TUNEL assay in comparison with only ligated hearts. These findings suggested that MSCs secrete paracrine acting materials with cardio-protective and/or inotropic properties, which may rescue hypoxic cardiomyocytes and improve ventricular functions via regulation of calcium influx.