--- - |2+ Abstract Aim Potassium channel accessory subunits (Kvβ) play a key role in cardiac electrical activity through ion channel modulation. In this study, we hypothesize that Kvβ2 regulates skeletal muscle growth and fibre phenotype via protein‐protein interactions. Methods Kvβ2 knockout mouse model was used for morphometric, immunohistochemical and biochemical analysis to evaluate the role of Kvβ2 in skeletal muscle physiology. Results Deletion of Kvβ2 gene in mice (Kvβ2 knockout, KO) leads to significant decrease in body weight along with skeletal muscle size. Key hindlimb muscles such as biceps, soleus and gastrocnemius were significantly smaller in size in KO mice compared to that of wild type. Morphometric measurements and histological analysis clearly point that the fibre size is decreased in each of the muscle type in KO compared with wild‐type mice. In addition, Kvβ2 deletion contributes to fibre‐type switching from fast to slow fibre as indicated by more abundant MHCI‐expressing fibres in gastrocnemius and soleus muscles, which may underscore the smaller muscle size alongside increase in U3 ubiquitin ligase; NEDD4 expression. Using targeted siRNA knockdown approach, we identified that Kvβ2 knockdown does not affect the myoblasts proliferation. However, Pax7 expression was significantly decreased in 4‐week‐old gastrocnemius muscle, suggesting that cellular reserve for growth may be deficient in KO mice. This is further supported by decreased migratory capacity of C2C12 cells upon siRNA‐targeted Kvβ2 knockdown. Conclusion Overall, this is the first report identifying that genetic deletion of Kvβ2 leads to decreased skeletal muscle size along with isotype switching. - Acta Physiologica, Volume 224, Issue 2, October 2018.