The accumulation of lipid at ectopic sites including the skeletal muscle and liver is a common consequence of obesity and is associated with tissue-specific and whole-body insulin resistance. Exercise is well known to improve insulin resistance by mechanisms not completely understood. We performed lipidomic profiling via mass spectrometry in liver and skeletal muscle samples from exercise trained mice, to decipher the lipid changes associated with exercise-induced improvements in whole body glucose metabolism. Obesity and insulin resistance was induced in C57BL/6J mice by high fat feeding for four weeks. Mice then underwent an exercise training program (treadmill running) five days a week (Ex) for four weeks or remained sedentary (Sed). Compared with Sed, Ex displayed improved (P<0.01) whole-body metabolism as measured via an oral glucose tolerance test. Deleterious lipid species such as diacylglycerol (DG) (P<0.05) and cholesterol esters (P<0.01) which accumulate with high fat feeding were decreased in the liver of trained mice. Furthermore, the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) (the PC/PE ratio), which is associated with membrane integrity and linked to hepatic disease progression was increased by training (P<0.05). These findings occurred without corresponding changes in the skeletal muscle lipidome. A concomitant decrease (P<0.05) was observed for the fatty acid transporters CD36 and FATP4 in the liver, suggesting exercise stimulates a coordinated reduction in fatty acid entry into hepatocytes. Given the important role of the liver in the regulation of whole body glucose homeostasis, hepatic lipid regression may be a key component by which exercise can improve metabolism.