Nemaline myopathy is the most common congenital myopathy and is notably caused by mutations in the ACTA1 gene encoding skeletal alpha-actin. The main features of ACTA1 null mutations (absence of skeletal alpha-actin) are generalized skeletal muscle weakness and premature death. A mouse model mimicking this condition can be rescued by incorporating the ACTC gene into skeletal muscles and hence, by overexpressing cardiac α-actin (ACTCCo/KO). Nevertheless, muscle fibres from ACTCCo/KO animals generate less force than normal cells (-20-25%). To understand the underlying mechanisms, here, we have undertaken a detailed functional study of fibres from ACTCCo/KO. Mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized fibres showed, upon maximal Ca2+ activation and under rigor conditions, lower stiffness but maintained meridional and equatorial reflections in ACTCCo/KO when compared with age-matched wild-type animals (WT). Overall, these results demonstrate that in ACTCCo/KO, the presence of cardiac alpha-actin instead of skeletal alpha-actin alters the formation of myosin cross-bridges by finely altering the strain of individual actomyosin interaction, thus lowering muscle fibre force production. These findings provide important information that has to be taken into consideration when considering the incorporation of the ACTC gene as a therapy for ACTA1-based nemaline myopathy.