Abstract Impairments in mitochondrial function and substrate metabolism are implicated in the etiology of obesity and type 2 diabetes. MicroRNAs (miRNAs) can degrade mRNA or repress protein translation and have been implicated in the development of such disorders. We used a contrasting rat model system of selectively bred high- (HCR) or low- (LCR) intrinsic running capacity with established differences in metabolic health to investigate the molecular mechanisms through which miRNAs regulate target proteins mediating mitochondrial function and substrate oxidation processes. Quantification of select miRNAs using the Rat miFinder miRNA PCR array revealed differential expression of 15 skeletal muscle (m. tibialis anterior) miRNAs between HCR and LCR rats (14 with higher expression in LCR; P<0.05). Ingenuity Pathway Analysis predicted these altered miRNAs to collectively target multiple proteins implicated in mitochondrial dysfunction and energy substrate metabolism. Total protein abundance of citrate synthase (CS; miR-19 target) and voltage-dependent anion channel 1 (miR-7a target) were higher in HCR compared to LCR cohorts (~57 and ~26%, respectively; P<0.05). A negative correlation was observed for miR-19a-3p and CS (r =0.32, P=0.015) protein expression. To determine if miR-19a-3p can regulate CS in vitro we performed luciferase reporter and transfection assays in C2C12 myotubes. MiR-19a-3p binding to the CS untranslated region did not change luciferase reporter activity, however miR-19a-3p transfection decreased CS protein expression (~70%; P<0.05). The differential miRNA expression targeting proteins implicated in mitochondrial dysfunction and energy substrate metabolism may contribute to the molecular basis mediating the divergent metabolic health profiles of LCR and HCR rats.