Skeletal muscle Na⁺,K⁺-ATPase plays a central role in the clearance of K⁺ from the extracellular fluid, therefore maintaining blood [K⁺]. Na⁺,K⁺-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high fat diet (HFD) and exercise training (ET) on skeletal muscle Na⁺,K⁺-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na⁺,K⁺-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days swim training in Wistar rats fed chow or HFD for 4 or 12 weeks. Skeletal muscle insulin resistance was observed after 12 weeks of HFD. Na⁺,K⁺-ATPase 1 subunit protein expression was increased 1.6-fold (p<0.05), while 2 and 1 subunits and protein expression was decreased 2-fold (p<0.01) in parallel with decrease in plasma membrane Na⁺,K⁺-ATPase activity after 4 weeks HFD. Exercise training restored 1, 2 and 1 subunit expression and Na⁺,K⁺-ATPase activity to control levels and reduced 2 subunit expression 2.2-fold (p<0.05). DNA binding activity of the 1 subunit-regulating transcription factor ZEB (AREB6) and 1 mRNA expression was increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of 2 and 1 subunit expression, was decreased after HFD. ET increased phosphorylation of the Na⁺,K⁺-ATPase regulatory protein phospholemman. Phospholemman mRNA and protein expression was increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the 2 subunit to plasma membrane was impaired by HFD, while 1 subunit translocation remained unchanged. Alterations in sodium pump function precede the development of skeletal muscle insulin resistance. Disturbances in skeletal muscle Na⁺,K⁺-ATPase regulation, particularly the 2 subunit, may contribute to impaired ion homeostasis in insulin resistant states such as obesity and Type 2 diabetes.