["Acta Physiologica, Volume 232, Issue 3, July 2021. ", "\nAbstract\n\nAim\nHighly prevalent diseases such as insulin resistance and heart failure are characterized by reduced metabolic flexibility and reserve. We tested whether Na/K‐ATPase (NKA)‐mediated regulation of Src kinase, which requires two NKA sequences specific to the α1 isoform, is a regulator of metabolic capacity that can be targeted pharmacologically.\n\n\nMethods\nMetabolic capacity was challenged functionally by Seahorse metabolic flux analyses and glucose deprivation in LLC‐PK1‐derived cells expressing Src binding rat NKA α1, non‐Src‐binding rat NKA α2 (the most abundant NKA isoform in the skeletal muscle), and Src binding gain‐of‐function mutant rat NKA α2. Mice with skeletal muscle‐specific ablation of NKA α1 (skα1−/−) were generated using a MyoD:Cre‐Lox approach and were subjected to treadmill testing and Western diet. C57/Bl6 mice were subjected to Western diet with or without pharmacological inhibition of NKA α1/Src modulation by treatment with pNaKtide, a cell‐permeable peptide designed by mapping one of the sites of NKA α1/Src interaction.\n\n\nResults\nMetabolic studies in mutant cell lines revealed that the Src binding regions of NKA α1 are required to maintain metabolic reserve and flexibility. Skα1−/− mice had decreased exercise endurance and mitochondrial Complex I dysfunction. However, skα1−/− mice were resistant to Western diet‐induced insulin resistance and glucose intolerance, a protection phenocopied by pharmacological inhibition of NKA α1‐mediated Src regulation with pNaKtide.\n\n\nConclusions\nThese results suggest that NKA α1/Src regulatory function may be targeted in metabolic diseases. Because Src regulatory capability by NKA α1 is exclusive to endotherms, it may link the aerobic scope hypothesis of endothermy evolution to metabolic dysfunction.\n\n"]