The increase in obesity rates in both children and adults in the United States may be a result of impaired or dysregulation of fat metabolism. Long chain acyl-CoA synthetases (ACSLs) may be key regulators of skeletal muscle cell fat metabolism, including fat oxidation and storage. The purpose of the study was to identify what ACSLs are present in human skeletal muscle, the effects of exercise on isoform abundance, and to see if a relationship exists between ACSLs and measures of fat metabolism. Enrolled participants (n=14) performed two metabolic study visits in a randomized, crossover design. During one visit the individuals remained sedentary (CON), while in the second, they performed a single session of moderate-intensity exercise (60 minutes at 65% VO2max). Fat oxidation was measured at rest and during exercise using indirect calorimetry. Muscle biopsies were taken after rest or exercise as well as two hours post exercise. We showed the presence of 4 of the 5 known ACSLs and a significant increase in ACSL 5 with exercise (P= 0.01). We also showed a relationship between ACSL1 and fat oxidation during exercise (P=0.07) and a significant correlation between ACSL6 and measures of triacylglycerol abundance (P= 0.05). We conclude that 4 of the 5 ACSL isoforms are present in human skeletal muscle with ACSL5 significantly affected by acute exercise training. We also conclude that ACSL1 during exercise and ACSL6 are related to measures of fat metabolism.