Neuropeptide Y (NPY) plays an important role in cardiovascular diseases including stress cardiomyopathy, hypertrophic cardiomyopathy, heart failure, diabetic cardiomyopathy, hypertension, and so on. However, inconsistent results related to the role of NPY in the different types of cardiomyopathies make the exact involvement of the peptide elusive. Considering these effects are known to be involved in energy balance, as the hearts energy producer, the mitochondria, should be investigated, and not only mitochondrial structure but also its potential. Up to now, the impact of NPY on energy metabolism and mitochondria in cultured neonatal rat cardiomyocytes has not been reported. The main objective of our study was to test the role of NPY in cultured neonatal rat cardiomyocytes. After 24-h stimulation of NPY, the ATP content and activity of the cardiomyocytes were determined by Cell Counting Kit-8 and ATP-dependent bioluminescence assay kit, respectively. To further measure these effects, mitochondrial membrane potential was measured by JC-1 staining, the change of mitochondrial structure was detected by transmission electron microscopy, and the levels of PGC-1α (a marker of mitochondrial energy metabolism) mRNA and protein expression were determined by real-time PCR and Western blotting, respectively. The results showed that after 24-h stimulation of NPY, ATP content and activity in the cardiomyocytes were decreased. Moreover, cardiomyocyte mitochondria were changed in morphology. Further, a decline of mitochondrial membrane potential was induced in a dose-dependent manner and the levels of PGC-1α mRNA and protein expression were up-regulated after being treated by different dose of NPY. The results indicate that energy metabolism is suppressed, mitochondrial structure and membrane potential damaged, and PGC-α is changed in cultured neonatal rat cardiomyocytes after being treated by NPY.