Background: Metabolic flexibility is the capacity of a system to adjust fuel (primarily glucose and fatty acids) oxidation based on nutrient availability. Pyruvate Dehydrogenase Kinase 4 (PDK4) is one of the main enzymes that play a critical role in metabolic flexibility. In current study, we examined PDK4 inhibition along with exercise training (ET) on the gene expression of Estrogen related-receptor alpha (ERRα ), medium-chain acyl-CoA dehydrogenase (MCAD), carnitine palmitoyl transferase-1b (CPT-1b), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α ), PDK4 and citrate synthase (CS) in skeletal muscle. Method: Sixty-four male Wistar rats (8 week-old) were randomly divided into 8 groups (n=8); 1-untreated control, 2-STZ-induced diabetic, 3-PDK4 inhibition, 4-endurance training (ET), 5-diabetic + PDK4 inhibition, 6-diabetic + ET, 7-PDK4 inhibition + ET, and 8-diabetic +ET + PDK4 inhibition. ERRα , MCAD, CPT-1b, PGC-1α , PDK4 and CS genes expressions were measured by Real-Time PCR and quantified by 2-Δ Δ Ct method. Results: ERRα , MCAD, CPT-1b, PGC-1α , PDK4, and CS expressions were significantly higher in non-diabetic+ Endurance Training group compared to the control group. The expressions of CPT-1b, MCAD and CS genes were significantly lower in the non-diabetic+ endurance training/PDK4 inhibition compared to the non-diabetic+ endurance training group, and the expressions of ERRα , CPT-1b and MCAD were significantly lower in the diabetic + PDK4 inhibition group compared to the diabetic group. Conclusion: In sum, PDK4 inhibition has negative effects on lipid metabolism in healthy rats, but in animals with diabetes, PDK4 inhibition can be used for improving lipid metabolism (over-expression of CS and PGC-1α ).