To understand the molecular mechanisms involved in the effect of exercise training, we examined hepatic transcriptional profiles using cDNA microarrays in exercise-trained and untrained mice with diet-induced obesity. C57BL/6J male mice (n = 10/group) were fed with a normal diet, high-fat diet (HFD), or HFD with exercise training for 12 weeks. The expression level of approximately 10,000 transcripts in liver tissues from each group was assessed using cDNA microarray analysis. Exercise training improved lipid profiles and hepatic steatosis and decreased body fat mass induced by the HFD. Seventy-three genes were differentially expressed in the HFD- and/or HFD with exercise training-treated groups, compared with the normal diet- and HFD-fed groups, respectively. Interestingly, the expression profiles involved in metabolism, such as elongation of very long chain fatty acids-like 2, lipin, and malic enzyme, were changed by exercise training. In addition, expression of genes altered by exercise training related to defense and stress response, including metallothionein 1 and 2 and heat shock protein, showed interesting findings. Our study showed beneficial effects of exercise training in preventing the development of obesity and metabolic disorders in mice with diet-induced obesity.