Tissue-engineered heart muscle may provide an alternative treatment modality for end-stage congestive heart failure. We have previously described a method to engineer contractile heart muscle in vitro (termed cardioids). This study describes a method to improve the contractile properties of cardioids utilizing thyroid hormone (T3) stimulation. Cardioids were engineered by promoting the self-organization of primary neonatal cardiac cells into a contractile tissue construct. Cardioids were maintained in standard cell culture media supplemented with varying concentrations of T3 in the range 1–5ng/ml. The contractile properties of the cardioids were evaluated 48h after formation. Stimulation with T3 resulted in an increase in the specific force of cardioids from an average value of 0.52 ± 0.16kPa (N = 6) for control cardioids to 2.42 ± 0.29kPa (N = 6) for cardioids stimulated with 3ng/ml T3. In addition, there was also an increase in the rate of contraction and relaxation in response to T3 stimulation. Cardioids that were stimulation with T3 exhibited improved pacing characteristics in response to electrical pacing at 1–5Hz and an increase in the degree of spontaneous contractility. Changes in the gene expression of SERCA2, phospholamban, α-myosin heavy chain, and β-myosin heavy chain correlated with the changes in contractile properties. This study demonstrates the modulation of the contractile properties of tissue-engineered heart muscle using T3 stimulation.