In this study, we wanted to verify whether the effect of insulin on calcium homeostasis depends on the heart's development stage. Using a quantitative 3D confocal microscopy, we tested the effect of a high insulin concentration (100 µU) in freshly cultured ventricular cardiomyocytes from newborn and adult rats. Our results showed that the cytosolic basal level of calcium was higher in newborn cardiomyocytes with no change in the nuclear basal calcium level compared with the adult cardiomyocytes; in addition, insulin induced a slow increase of cytosolic and nuclear calcium in newborn ventricular cardiomyocytes, followed by two phases. However, the first phase of slow cytosolic and nuclear calcium increase was absent in adult rat ventricular cardiomyocytes. Furthermore, the time to the onset of increase of cytosolic and nuclear calcium was longer in newborn cardiomyocytes compared with adults. Moreover, the time to peak of the calcium transient was shorter in newborns than in adult cardiomyocytes. These results demonstrate that insulin differently regulates calcium homeostasis in newborns than in adult cardiomyocytes. Thus, newborn rat cardiomyocytes, commonly used in research as a model for adult cardiomyocytes, should be used with caution when dealing with insulin in normal and disease conditions.