Identification of factors regulating cardiomyocyte survival and growth is important to understand the pathogenesis of congenital heart diseases. Little is known about the molecular mechanism of cardiac functions triggered by serotonin. The link between signaling circuitry of external stimuli and the mitochondrial apoptotic machinery is of wide interest in cardiac diseases. Using cultured cardiomyocytes and 5-hydroxytryptamine (5-HT)2B-receptor knockout mice as an animal model of dilated cardiomyopathy, for the first time we show that serotonin via the Gq-coupled 5-HT2B-receptor protect cardiomyocytes against serum deprivation-induced apoptosis as manifested by DNA fragmentation, nuclear chromatin condensation, and TUNEL labeling. Serotonin prevents cytochrome c release and caspase-9 and -3 activation after serum deprivation via cross-talks between phosphatidylinositol-3 kinase/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Serotonin binding to 5-HT2B-receptor activates ERK kinases to inhibit Bax expression induced by serum deprivation. Serotonin via phosphatidylinositol-3 kinase/Akt can activate NF-kappaB that is required for the regulation of the mitochondrial adenine nucleotide translocator (ANT-1). Parallel to these observations, ultrastructural analysis in the 5-HT2B-receptor knockout mice heart revealed pronounced mitochondrial defects in addition to altered mitochondrial enzyme activities (cytochrome oxidase and succinate dehydrogenase) and ANT-1 and Bax expressions. These findings identify 5-HT as a novel survival factor targeting mitochondria in cardiomyocytes.