More than one‐third of adults and 17% of children in the United States are classified as obese. One contributor to the development of obesity is an increase energy intake accompanied by a decrease energy expenditure. Muscle plays an important role in energy utilization and production in human body. Excessive fat mass accumulation in muscle blunts energy production such as fatty acid oxidation. The objectives of this study were to: 1) determine if the branched‐chain amino acid leucine lowers intracellular fatty acid uptake, and 2) determine if mitochondria are involved in the leucine‐mediated effect on fatty acid uptake. C2C12 myotubes were used as a cell model for muscle. Cells were treated with palmitate for 24 hours in order create an obesogenic muscle model. All cells were serum and leucine starved for 24 hours before receiving one of the following four treatments: control (CON), palmitate (PALM, palmitate treated for 24h), palmitate + 0.5mM leucine (PALM+0.5mM LEU,, palmitate and 0.5mM leucine co‐treated for 24h), palmitate + 1.5mM leucine (PALM+1.5mM LEU,, palmitate and 1.5mM leucine co‐treated for 24h). Intracellular lipid deposition was measured using oil red o staining kit and quantified using ImageJ software. Both doses of leucine showed significant effect (p<0.05) on lowering the intake of intracellular lipid uptake in PALM treated cells. Real‐time PCR results showed higher (p<0.05) expression of uncoupling proteins (UCP2 and UCP3), which are involved in ATP synthesis, in both leucine treatment groups compared with PALM. There was no significant difference in gene expression of NRF1, SIRT1, Tfam, PPARγ and ANT1, all involved in mitochondria DNA transcription. In conclusion, these data suggest leucine promotes energy metabolism through regulating uncoupling protein in mitochondria in C2C12 myotubes, indicating a possible target for improving obesity.Support or Funding InformationArkansas Biosciences InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.