Growing three-dimensional scaffolds that contain more than a few layers of seeded cells is crucial for the creation of thick and viable cardiac tissues. To achieve this goal, a bioengineered cardiac patch (the MSC patch) composed of a sliced porous biological scaffold inserted with multilayered mesenchymal stem cells (MSCs) was developed for myocardial repair in a syngeneic rat model. After culture, sliced layers of the scaffold were stuck together and seeded MSCs were redistributed throughout the scaffold. Immunofluorescence analyses indicated that cells were viable and tightly adhered to a robust fibronectin meshwork within the scaffold. Results of echocardiography and heart catheterization revealed that the MSC-patch group had a superior heart function to the infarct group. Cells together with neo-muscle fibers and neo-microvessels were clearly observed in the entire MSC patch to fill its original pores, indicating that the implanted patch became well integrated into the host. The thickness of the retrieved MSC patch increased significantly as compared to that before implantation. When compared with the infarct group, expressions of angiogenic cytokines (bFGF, vWF and PDGF-B) and cardioprotective factors (IGF-1 and HGF) were significantly increased in the MSC-patch group. The aforementioned results indicated that transplantation of the MSC patch could restore the dilated LV and preserve cardiac functions after infarction.