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) by treating newborn pups (P0-1) with 6-hydroxydopamine (6-OHDA). Immunofluorescence staining of tyrosine hydroxylase, a marker of sympathetic neurons, demonstrated that the sympathetic neurons were almost completely eliminated in P7 to 21 mice hearts by 6-OHDA treatment. 6-OHDA treatment significantly reduced left ventricular contractility compared with the control at P21. However, neither L type calcium channel activity nor calcium transients evoked by field stimulation were unchanged in the isolated cardiomyocytes from P21 mice hearts. Fluorescent imaging of T-tubule structures demonstrated that cardiomyocytes from 6-OHDA-treated mice hearts have reduced regularity of T-tubule structure. Furthermore, the protein abundance of alpha-SMA, a vascular smooth muscle cell marker, was significantly reduced in 6-OHDA-treated mice hearts at P21. These results suggest that sympathetic innervation is likely to play critical roles in the postnatal maturation of cardiomyocytes and coronary circulation.