Mitochondrial Ca(2+) is known to change dynamically, regulating mitochondrial as well as cellular functions such as energy metabolism and apoptosis. The NCLX gene encodes the mitochondrial Na(+)-Ca(2+) exchanger (NCXmit), a Ca(2+) extrusion system in mitochondria. Here we report that the NCLX regulates automaticity of the HL-1 cardiomyocytes. NCLX knockdown using siRNA resulted in the marked prolongation of the cycle length of spontaneous Ca(2+) oscillation and action potential generation. The upstrokes of action potential and Ca(2+) transient were markedly slower, and sarcoplasmic reticulum (SR) Ca(2+) handling were compromised in the NCLX knockdown cells. Analyses using a mathematical model of HL-1 cardiomyocytes demonstrated that blocking NCXmit reduced the SR Ca(2+) content to slow spontaneous SR Ca(2+) leak, which is a trigger of automaticity. We propose that NCLX is a novel molecule to regulate automaticity of cardiomyocytes via modulating SR Ca(2+) handling.