The half-sarcomere, the functional unit of skeletal muscle, is able to produce power at high efficiency or resist a sudden increase in load with low metabolic cost, due to the combination of the function of the myosin II motors in the thick filament overlapped with the thin filament and the meshwork of cytoskeleton proteins acting as a scaffold. The importance of cytoskeleton proteins is illustrated by the identification of mutations in many of the corresponding human genes in patients with skeletal myopathies. Here we studied the role of myopalladin (MYPN), a protein located in the Z-line and the I-band. MYPN gene mutations have been identified in patients with limb-girdle dystrophy as well as dilated, hypertrophic and restrictive cardiomyopathy (Duboscq-Bidot et al. Cardiovasc Res 77:118, 2008; Purevjav et al. Hum Mol Genet 21:2039, 2012). To provide insights into the physiological role of this protein and the mechanisms leading to myopathy, the mechanical performances of skeletal muscles from wt and KO mice were determined. In EDL muscle, the absence of MYPN (i) decreases the isometric force (T0) by 48% and the cross-sectional area (CSA), calculated by the muscle wet weight, by 21%; (ii) decreases the power at any load and (iii) does not affect the curvature of the force-velocity relation. In skinned fibers from the same muscles T0 is reduced in proportion to CSA, indicating that the CSA of EDL muscle is overestimated in KO mice with respect to control. Thus the reduced muscle performance in KO mice is due to the reduction in fiber dimension while the kinetics of actin-myosin interaction is unaffected. Injection of an adeno-associated virus (AAV) vector expressing the wt form of MYPN results in a substantial rescue of muscle performance. Supported by Ministero della Salute, Telethon and MIUR (Italy).