Accumulation of triacylglycerols (TAGs) and acylcarnitines in skeletal muscle upon high-fat (HF) feeding is the resultant of fatty acid uptake and oxidation and is associated with insulin resistance. As medium-chain fatty acids (MCFAs) are preferentially β-oxidized over long-chain fatty acids, we examined the effects of medium-chain TAGs (MCTs) and long-chain TAGs (LCTs) on muscle lipid storage and whole-body glucose tolerance. Rats fed a low-fat (LF), HFLCT, or an isocaloric HFMCT diet displayed a similar body weight gain over 8 weeks of treatment. Only HFLCT increased myocellular TAG (42.3 ± 4.9, 71.9 ± 6.7, and 48.5 ± 6.5 µmol/g for LF, HFLCT, and HFMCT, respectively, P < 0.05) and long-chain acylcarnitine content (P < 0.05). Neither HF diet increased myocellular diacylglycerol (DAG) content. Intraperitoneal (IP) glucose tolerance tests (1.5 g/kg) revealed a significantly decreased glucose tolerance in the HFMCT compared to the HFLCT-fed rats (802 ± 40, 772 ± 18, and 886 ± 18 area under the curve for LF, HFLCT, and HFMCT, respectively, P < 0.05). Finally, no differences in myocellular insulin signaling after bolus insulin injection (10 U/kg) were observed between LF, HFLCT, or HFMCT-fed rats. These results show that accumulation of TAGs and acylcarnitines in skeletal muscle in the absence of body weight gain do not impede myocellular insulin signaling or whole-body glucose intolerance.