LKB1 phosphorylates members of the AMP-activated protein kinase (AMPK) family. LKB1 and AMPK in the skeletal muscle are believed to regulate not only fuel oxidation during exercise but also exercise capacity. LKB1 was also required to prevent diaphragm fatigue, which was shown to affect exercise performance. Using mice expressing dominant negative (DN) mutants of LKB1 and AMPK, specifically in the skeletal muscle but not in the heart, we investigated the roles of LKB1 and AMPK activity in exercise performance and the effects of these kinases on the characteristics of respiratory and locomotive muscles. In the diaphragm and gastrocnemius, both AMPK-DN and LKB1-DN mice showed complete loss of AMPKα2 activity, and LKB1-DN mice showed a reduction in LKB1 activity. Exercise capacity was significantly reduced in LKB1-DN mice, with a marked reduction in oxygen consumption and carbon dioxide production during exercise. The diaphragm from LKB1-DN mice showed an increase in myosin heavy chain IIB and glycolytic enzyme expression. Normal respiratory chain function and CPT I activity were shown in the isolated mitochondria from LKB1-DN locomotive muscle, and the expression of genes related to fiber type, mitochondria function, glucose and lipid metabolism, and capillarization in locomotive muscle was not different between LKB1-DN and AMPK-DN mice. We concluded that LKB1 in the skeletal muscle contributes significantly to exercise capacity and oxygen uptake during exercise. LKB1 mediated the change of fiber-type distribution in the diaphragm independently of AMPK and might be responsible for the phenotypes we observed.