We provide evidence that cell anchorage to the fibrin-based 3D environment has profound effects on cell spreading, actin cytoskeleton organization and nuclear shape of human myoblasts. Moreover, our results revealed complex interactions between muscle cells and their surrounding matrix that are of critical importance for pathophysiological applications of 3D muscle culture and for the engineering of a functional skeletal muscle. Because the cytoskeleton network is required for cell spreading, the architecture of the actin cytoskeleton was analyzed in myoblasts embedded. The elastic modulus of the gel further increased upon myotube differentiation, further supporting the role of inherent cell contractility in the modulation of the fibrin gel stiffness. These findings do not exclude the possibility that factors secreted by myoblasts and/or myotubes into the ECM also modulated the scaffold stiffness during culture, a point that deserves further investigation. Changes in scaffold stiffness have important implications for engineered muscle tissue given that sarcomeres will not form in cultured myocytes unless they grow on a substrate with a stiffness at least equal to physiological muscle stiffness, i.e. value higher than that of fibrin alone. Therefore, the capacity of myoblasts to stiffen the fibrin scaffold during the time course of differentiation appears crucial to achieve functional engineered muscle tissue. In addition, it is noteworthy that the final elastic modulus of our construct matched the stiffness of normal skeletal muscle, further supporting the formation of a contractile engineered muscle tissue. However, the fact that the expression of adhesion proteins was higher in 3D than in 2D cultures could be related to their more uniform distribution at the cell membrane, thus facilitating cell anchorage to the matrix. Interestingly, we reported dramatically transformed nuclear morphology in myocytes cultured in 3D. Thicker and more elongated nuclei observed in our 3D matrix were reminiscent of those physiologically observed in the skeletal muscle tissue. The precise mechanisms which regulate the shape of the nucleus are not yet fully understood. It has been shown that nuclear shape is at least in part modulated by the interactions between the cytoskeleton and the ‘‘linkers of the nucleoskeleton to the cytoskeleton’’ complex that spans the nuclear envelope and in turn anchor networks of filaments to the nucleus. In mouse embryonic fibroblasts cultured on stiff 2D surface, actin has been proposed to form an apical cap to the nucleus that modulates the shape of the nucleus. Disruption of this cap directly or through rupture of the LINC complex increases the thickness of the nucleus. In our cells grown in 3D, actin stress fibers were found around the nucleus, indicating that the presence of an actin cap was not per se the unique determinant of the shape of the nucleus. Finally, the presence of actin stress fibers both at the apical and basal sides of the nucleus in human myocytes cultured on 2D may indicate that the perinuclear cap distribution of actin may be cell- and/or species-specific. In conclusion, our results showed that myoblasts embedded in a fibrin matrix demonstrate mechanotransductive responses by changing the organization of the adhesion complex, the actin cytoskeleton and the shape of the nucleus. This complex myocyte behavior is of critical importance for engineering a functional skeletal muscle tissue and for pathophysiological applications of engineered muscle tissues.