Fatty acid oxidation is highly regulated in skeletal muscle and involves several sites of regulation, including the transport of fatty acids across both the plasma and mitochondrial membranes. Transport across these membranes is recognized to be primarily protein mediated, limited by the abundance of fatty acid transport proteins on the respective membranes. In recent years, evidence has shown that fatty acid transport proteins move in response to acute and chronic perturbations; however, in human skeletal muscle the localization of fatty acid transport proteins in response to training has not been examined. Therefore, we determined whether high-intensity interval training (HIIT) increased total skeletal muscle, sarcolemmal, and mitochondrial membrane fatty acid transport protein contents. Ten untrained females (22 ± 1 yr, 65 ± 2 kg; V̇o2peak: 2.8 ± 0.1 l/min) completed 6 wk of HIIT, and biopsies from the vastus lateralis muscle were taken before training, and following 2 and 6 wk of HIIT. Training significantly increased maximal oxygen uptake at 2 and 6 wk (3.1 ± 0.1, 3.3 ± 0.1 l/min). Training for 6 wk increased FAT/CD36 at the whole muscle (10%) and mitochondrial levels (51%) without alterations in sarcolemmal content. Whole muscle plasma membrane fatty acid binding protein (FABPpm) also increased (48%) after 6 wk of training, but in contrast to FAT/CD36, sarcolemmal FABPpm increased (23%), whereas mitochondrial FABPpm was unaltered. The changes on sarcolemmal and mitochondrial membranes occurred rapidly, since differences (≤2 wk) were not observed between 2 and 6 wk. This is the first study to demonstrate that exercise training increases fatty acid transport protein content in whole muscle (FAT/CD36 and FABPpm) and sarcolemmal (FABPpm) and mitochondrial (FAT/CD36) membranes in human skeletal muscle of females. These results suggest that increases in skeletal muscle fatty acid oxidation following training are related in part to changes in fatty acid transport protein content and localization.