Upon intramuscular entry, fatty acids are converted to amphiphatic fatty acyl-CoAs by action of the acyl-CoA synthetase (ACS) enzymes. While it has been reported that insulin resistant skeletal muscle shows an accumulation of fatty acyl-CoAs, the role of the enzymes which catalyze their synthesis is still sparsely studied in human muscle, in particular the influence of obesity, and insulin resistance. We analyzed muscle biopsies obtained from normal weight controls (n = 7, average BMI 24), males/females with obesity (n = 7, average BMI 31), and males/females with obesity and type 2 diabetes (T2D) (n = 7, average BMI 34), for relevant ACS (long-chain acyl-CoA synthetase 1 (ACSL1), −3 (ACSL3) and − 4 (ACSL4), fatty acid transport protein 1 (FATP1) and − 4 (FATP4)). The mRNA expression was determined by real-time PCR, and total oleoyl-CoA synthetase activity was measured. In the males/females with obesity and T2D, the response to 16 weeks of exercise training with minor weight loss was evaluated. ACSL1 is the dominantly expressed ACS isoform in human skeletal muscle. The content of total ACS mRNA, as well as ACSL1 mRNA, were lower in muscle of males/females with obesity and T2D. Exercise training in the males/females with obesity and T2D increased the total ACS enzyme activity, along with a lowering of the HOMA-IR index. The capacity for synthesis of fatty acyl-CoAs is lower in skeletal muscle of obese males/females with T2D. This suggests a decreased ability to convert fatty acids to fatty acyl-CoAs, which in turn may affect their entry into storage or metabolic pathways in muscle. Thus, the accumulation of fatty acyl-CoAs in the obese or insulin resistant state that has been shown in previous reports is not likely to result from increased fatty acid acylation. The upregulation of ACS activity by exercise training appears beneficial and occurred concomitantly with increased insulin sensitivity.