Cell transplantation is a promising approach for patients with myocardial infarction. However, following injection, retention of the transplanted cells in the injected area remains a central issue, which can be deleterious to cell transplantation therapy. We hypothesized that the use of cell sheet fragments, with the preservation of extracellular matrix (ECM), may significantly increase cell retention and thus improve cell therapy. Mesenchymal stem cell (MSC) sheet fragments with ECM were fabricated. Experimental myocardial infarction was created in male syngeneic Lewis rats. Thirty minutes after myocardial infarction, an intramyocardial injection was conducted with a needle directly into the peri-infarct areas. There were four treatment groups (n > or = 10): sham; phosphate buffered saline; dissociated MSCs; and MSC sheet fragments. Echocardiography and pressure measurements were assessed postoperatively. At retrieval, the hearts were fixed for histological evaluation. After injection, the MSC sheet fragments remained intact, while the complete cell sheets were torn into pieces. The results obtained in the echocardiography and pressure measurements revealed a superior heart function in the MSC sheet fragment group compared with the dissociated MSC group (P < 0.05). The MSC sheet fragments were able to conform and align their inherent ECM along the interstices of the muscular tissues at the injection sites, while only a few cells were identified in the dissociated MSC group at 12 weeks postoperatively. Additionally, transplantation of the MSC sheet fragments stimulated a significant increase in vascular density (P < 0.05) and enhanced the graft/host cell connection. The MSC sheet fragments may serve as a cell delivery vehicle by providing a favourable ECM environment to retain the transplanted cells and improve the efficacy of therapeutic cell transplantation.