Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSC) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here, we developed a new platform that combines three-dimensional cell cultivation in a microfabricated system with electrical stimulation to mature hPSC-derived cardiac tissues. We utilized quantitative structural, molecular and electrophysiological analyses to elucidate the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allowed for the generation of 3-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation markedly increased myofibril ultrastructural organization, displayed elevated conduction velocity and altered both the electrophysiological and Ca2+ handling properties versus non-stimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.