Forces generated by heart muscle contraction must be balanced by adhesion to the extracellular matrix (ECM) and to other cells for proper heart function. Decades of data have suggested that cell-ECM adhesions are important for sarcomere assembly. However, the relationship between cell-ECM adhesions and sarcomeres assembling de novo remains untested. Sarcomeres arise from muscle stress fibers (MSFs) that are translocating on the top (dorsal) surface of cultured cardiomyocytes. Using an array of tools to modulate cell-ECM adhesion, we established a strong positive correlation between the extent of cell-ECM adhesion and sarcomere assembly. On the other hand, we found a strong negative correlation between the extent of cell-ECM adhesion and the rate of MSF translocation, a phenomenon also observed in nonmuscle cells. We further find a conserved network architecture that also exists in nonmuscle cells. Taken together, our results show that cell-ECM adhesions mediate coupling between the substrate and MSFs, allowing their maturation into sarcomere-containing myofibrils.