In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analyzed the small-angle X-ray diffraction patterns of isolated membrane-permeabilized myofibers. Results showed that upon addition of Ca²⁺, the intensity changes of the second (1/19nm^-1) and sixth (1/5.9nm^-1) actin layer lines and of the first myosin meridional reflection (1/14.3nm^-1) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6μm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length>3.6μm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction.