We have developed an efficient decellularization process for the isolation of extracellular matrix (ECM) from native cardiac tissue. The isolated ECM exhibited desirable mechanical properties in terms of elasticity, strength and durability-properties required from scaffolds used for cardiac tissue repair. This study further investigates the potential use of this scaffold for cardiac tissue engineering in terms of interactions with seeded cells and biocompatibility. We used the commonly studied fibroblasts, cardiomyocytes, and mesenchymal stem cells, which were isolated and seeded onto the scaffold. Cell density and distribution were followed by 3,3'-dioctadecyloxacarbocyanine perchlorate staining, and their proliferation and viability were assessed by AlamarBlue® assay and fluorecein-diacetate/propidium iodide staining. Fibroblast-seeded scaffolds shrank to 1-2mm3 spheroids, and their glycosaminoglycans significantly increased by 23%. The expression of ECM remodeling-related mRNAs of collagens I and III, matrix metalloproteinase 2, and type 1 tissue inhibitor of metalloproteinases was quantified by real-time polymerase chain reaction, and was found significantly elevated in fibroblast-seeded scaffold, compared with the control cells on plates. Fibroblast-seeded scaffolds lost some flexibility, yet gained strength compared with the acellular scaffolds, as shown by mechanical testing. Scaffold seeded with cardiomyocyte began to beat in concert few days after seeding, and the myocytes expressed typical functional cardiac markers such as α-actinin, troponin I, and connexin43. The cells revealed aligned elongated morphology, as presented by immunofluorescent staining and scanning electron microscopy. Mesenchymal stem cell-seeded scaffolds maintained viability over 24 days in culture. These findings further strengthen the potential use of acellular cardiac ECM as a biomaterial for heart regeneration.