The sympathetic nervous system is important for the adult heart to adapt to the changes in environmental stresses. Besides that, it has been suggested that sympathetic innervation affects postnatal development of the heart. Several lines of evidence have indicated that sympathetic innervation changes the modes of cardiomyocyte growth from proliferation to hypertrophy and the membrane excitability by altering ion channel activities. However, most of these findings were obtained from the in vitro co-culture of neonatal cardiomyocytes and sympathetic neurons. Therefore, whether it is also the case in vivo remains largely unknown. In this study, we have analyzed the effect of chemical sympathetic denervation on the postnatal murine heart development before weaning (at postnatal day (P) ~20). Six-hydroxydopamine (6-OHDA) is an analog of dopamine which is selectively transported into and kills sympathetic neurons by releasing reactive oxygen species. The newborn pups (P0-1) were treated with 6-OHDA. Immunofluorescence staining of tyrosine hydroxylase, a marker of sympathetic neurons, demonstrated that the heart was almost completely sympathetic denervated from P7 to 21. Under this condition, ventricular L-type calcium channel activity and contractility were significantly reduced compared with the control at P21. These data suggest that sympathetic innervation plays critical roles in the postnatal heart development.