Abstract Body: Background: The postnatal mammalian heart undergoes a major metabolic shift, transitioning from anaerobic glycolysis to aerobic fatty acid β-oxidation (FAO) as it matures. This shift depends critically on the energy produced from specific substrates by developing mitochondria (Mt). Hypothesis: We hypothesize that precise temporal sequence of metabolic changes during the first two weeks of the heart is determined by substrate specific mitochondrial maturation. Objective: To performmetabolic profiling of mouse hearts at two-day intervals during the critical postnatal (P) cardiac maturation window (P2-P14 days). Methods: Hearts from C57BL/6J mouse were collected between P2 and P14 (N=3/timepoint), snap-frozen, and stored at –80°C. These hearts were subjected to metabolomics by extracting the metabolites from 50–80mg of tissues and analyzed via RP-UPLC coupled Orbitrap/Q-Exactive mass spectrometer. Mt-maturation was assessed by measuring biogenesis by assessing the contents of mt proteins, (immunoblotting of proteins), mtDNA content and gene expression by qRT-PCR , and Fe-S cluster content by EPR spectroscopy . We employed Welch's t-test for paired comparisons and PCA/two-way ANOVA for multiple comparisons. Results: Long-chain (Lc-) acylcarnitines (C18, C16, and C16:1) were significantly abundant in P2–P14 (P<0.05) hearts. Specifically, palmitoylcarnitine (C16) increased sharply between P6–P8, while stearoylcarnitine (C18) increased gradually during P2–P14. Palmitoleoylcarnitine (C16:1) showed variable expression independent of specific age. The P6–P8 metabolic transition coincided with increased expression of mitochondrial biogenesis markers(PPARα, CPT1B, and ACADVL), suggesting acylcarnitines drive the metabolic shift in maturing hearts. This was evidenced by concurrent decreases in glycolytic markers (Hexokinase-1 and GLUT1/4) and mt-DNA copy numbers. Iron-sulfur clusters content showed a marked increase in mt, from P8, confirming enhanced activity to meet the energetic demands of cardiac maturation. Conclusion: Cardiac energy metabolism undergoes an abrupt transition from glycolysis to FAO between P6-P7, primarily regulated by Lc-acylcarnitines alongside driven by mt maturation. Therefore, palmitoylcarnitine precise metabolism appears to be important for balancing energy production during postnatal cardiac development.