A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by carnitine palmitoyltransferase 1 (CPT1). To investigate the role of CPT1 in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT1 in muscle of rodents. A vector expressing the human muscle isoform of CPT1 was electrotransferred into the right lateral muscles of the distal hind limb (tibialis cranialis (TC) and extensor digitorum longus (EDL)) of rats and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT1 protein expression peaked 7 days after IVE (+104%, P<0.01). This was associated with an increase in maximal CPT1 activity (+30%, P<0.001) and a similar increase in palmitoyl CoA oxidation (+24%; p<0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium chain FA, octanoyl CoA, and CPT1 sensitivity to inhibition by malonyl CoA were not altered by CPT1 overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT1 overexpression (+28%, P<0.01), while FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (-17%, P<0.01). As a result intramyocellular TAG content was decreased with CPT1 overexpression in both the TC (-25%, P<0.05) and the EDL (-45%, P<0.05). These studies demonstrate that acute overexpression of CPT1 in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT1 in regulating muscle FA metabolism.