Carnitine acetyltransferase (CRAT) deficiency has previously been shown to result in muscle insulin resistance due to accumulation of long-chain acylcarnitines. However, differences in the acylcarnitine profile and/or changes in gene expression and protein abundance of CRAT in myotubes obtained from obese patients with type 2 diabetes mellitus (T2DM) and glucose-tolerant obese and lean controls remain unclear. The objective of the study was to examine whether myotubes from obese patients with T2DM express differences in gene expression and protein abundance of CRAT and in acylcarnitine species pre-cultured under glucose and insulin concentrations similar to those observed in healthy individuals in the over-night fasted, resting state. Primary myotubes obtained from obese persons with or without T2DM and lean controls (n=9 in each group) were cultivated and harvested for LC-MS-based profiling of acylcarnitines. The mRNA expression and protein abundance of CRAT were determined by qPCR and Western Blotting, respectively. Our results suggest that the mRNA levels and protein abundance of CRAT were similar between groups. Of the 14 different acylcarnitine species measured by LC-MS, the levels of palmitoylcarnitine (C16) and octadecanoylcarnitine (C18) were slightly reduced in myotubes derived from T2DM patients (p<0.05) compared to glucose-tolerant obese and lean controls. This suggests that the CRAT function is not the major contributor to primary insulin resistance in cultured myotubes obtained from obese T2DM patients.