<p>The human myocardium is a mechanically active tissue whose anisotropic organization is crucial for proper function. Disruption of this organization after injury contributes to adverse remodeling and heart failure. While mechanobiological phenomena like strain avoidance and contact guidance have been used to promote anisotropy, the role of contraction-induced strain – driven by cardiomyocytes – has been largely overlooked. To investigate this, we engineered cardiac microtissues by embedding cardiac cells in constrained collagen hydrogels, modeling aligned (anisotropic) and disorganized (isotropic) tissues. By varying tissue constraint geometry, we created conditions that either promote or restrict anisotropy and assessed the contribution of cardiomyocyte contraction. Using combined in vitro experiments and in silico modeling, we demonstrate that constraint geometry influences tissue anisotropy and that cardiomyocyte contraction enhances this effect via cell-mediated collagen prestretch. Furthermore, establishing anisotropy in contracting microtissues by manipulating mechanical constraints significantly improves sarcomere development and overall tissue contractility. Our approach thus provides evidence of the functional benefits of beating cardiomyocytes in restoring cardiac tissue anisotropy. Additionally, our study offers insights into the dynamic interplay between tissue contractility, mechanical tension, and organization, presenting potential pathways for cardiac regenerative strategies by actively harnessing contraction-induced matrix remodeling to guide tissue architecture and function.</p>