Insulin is a hormone that plays an important role in the regulation of the metabolism of all the main nutrients. Its main function is to stimulate glucose uptake and disposal or utilization by the cells and thus to decrease blood glucose concentration. However, it also inhibits breakdown of proteins and lipids and promotes their synthesis. Type 1 diabetes is a disease in which insulin secretion is impaired because of destruction of pancreatic β-cells. It is characterized by hyperglycemia and increased reliance on fat oxidation. This is seen also as altered gene expression patterns. The purpose of this study was to look into the effects of insulin deficiency on exercise-induced acute responses in the expression of genes and the activation of signaling pathways involved in fatty acid oxidation. Male NMRI mice (n = 64) were randomly assigned into three streptozotocin-induced diabetic and three healthy groups. Two healthy and two diabetic groups performed a single one-hour bout of treadmill running (21 m/min, 2.5o incline) and were sacrificed either three or six hours after exercise. Gastrocnemius muscles were dissected and mRNA expression and protein expression and phosphorylation were analyzed with RT-PCR and Western blotting respectively. PGC-1α mRNA expression increased (p < 0.001) after exercise in both healthy and diabetic mice, but the response was higher in diabetic mice (p < 0.05). PDK4 mRNA expression increased after exercise only in diabetic mice (p < 0.05). Diabetic mice showed a more pronounced response in CPT1B mRNA six hours after exercise compared with healthy exercised mice (p < 0.05). Contrary to mRNA level results, PGC-1α protein content did not change in response to exercise when compared with sedentary counterparts of the same health status. The analysis of AMPK and p38 MAPK phosphorylation did not suggest activation of these pathways in response to exercise. Even a decrease in AMPK phosphorylation was seen six hours after exercise in healthy mice (p < 0.05) while p38 MAPK phosphorylation was de-creased in diabetic mice three hours after exercise (p < 0.05). There were no significant changes in proteins PDK4, CPT1B, sirtuins 1, 3 and 6, ACC or Cyt c. These results suggest that diabetic mice have more pronounced exercise-induced responses in the expression of genes related to increased fatty acid oxidation. These changes may be mediated by in-creased PGC-1α activation as no increases were seen in PGC-1α protein expression. The time points were not optimal for protein level analyses and thus, further studies are needed to clarify protein phosphorylation and expression changes and to find out whether the gene expression changes are reflected to the level of substrate metabolism.