Dietary trans-fatty acids are associated with increased risk of cardiovascular disease and have been implicated in the incidence of obesity and type 2 diabetes mellitus (T2DM). It is established that high-fat saturated diets, relative to low-fat diets, induce adiposity and whole-body insulin resistance. Here, we test the hypothesis that markers of an obese, prediabetic state (fatty liver, visceral fat accumulation, insulin resistance) are also worsened with provision of a low-fat diet containing elaidic acid (18:1t), the predominant trans-fatty acid isomer found in the human food supply. Male 8-week-old Sprague-Dawley rats were fed a 10% trans-fatty acid enriched (LF-trans) diet for 8 weeks. At baseline, 3 and 6 weeks, in vivo magnetic resonance spectroscopy (1H-MR) assessed intramyocellular lipid (IMCL) and intrahepatic lipid (IHL) content. Euglycemic-hyperinsulinemic clamps (week 8) determined whole-body and tissue-specific insulin sensitivity followed by high-resolution ex vivo 1H-NMR to assess tissue biochemistry. Rats fed the LF-trans diet were in positive energy balance, largely explained by increased energy intake, and showed significantly increased visceral fat and liver lipid accumulation relative to the low-fat control diet. Net glycogen synthesis was also increased in the LF-trans group. A reduction in glucose disposal, independent of IMCL accumulation was observed in rats fed the LF-trans diet, whereas in rats fed a 45% saturated fat (HF-sat) diet, impaired glucose disposal corresponded to increased IMCLTA. Neither diet induced an increase in IMCLsoleus. These findings imply that trans-fatty acids may alter nutrient handling in liver, adipose tissue, and skeletal muscle and that the mechanism by which trans-fatty acids induce insulin resistance differs from diets enriched with saturated fats.