The aim of this study was to investigate whether Cyclosporin-A (CsA)-induced myocardial injury is mediated by elevating the intracellular calcium concentration ([Ca2+]i) through the Calcium sensing receptor (CaSR). Cultured neonatal rat cardiomyocytes were treated with CsA, with or without pretreatment with the CaSR-specific antagonist NPS2390 or the CaSR-specific agonist gadolinium chloride (GdCI3). At 2 h, 4 h, 6 h and 8 h after CsA treatment, the ultrastructural changes of the cardiomyocytes were observed. In addition, the lactate dehydrogenase (LDH) and creatine kinase (CK) release from the cardiomyocytes, the [Ca2+]i and the level of CaSR expression were determined. With increasing time of CsA treatment, ultrastructural damage of cardimyocytes gradually aggrevated, LDH and CK release and [Ca2+]i also gradually increased. CaSR mRNA and protein expression increased at 4 h after CsA treatment. Compared with CsA treatment alone, pretreatment with NPS2390 lessened the ultrastructural damage of the cardiomyocytes as well as decreased the LDH and CK release, [Ca2+]i and the expression of the CaSR mRNA and protein. Conversely, pretreatment with GdCI3 aggravated the ultrastructural damage of the cardiomyocytes as well as increased LDH and CK release, [Ca2+]i and the expression of the CaSR mRNA and protein. These results demonstrate that CsA induced cardiomyocyte injury in a time-dependent manner. Moreover, CsA-induced cardiomyocyte injury was related to CaSR-mediated intracellular calcium overload. These findings provide new insight into the mechanisms involved in CsA-induced myocardial injury.