Cell transplantation via local intramuscular injection is a promising therapy. However, the cells used are usually expanded in vitro and dissociated by trypsinization, which may be harmful to the cells. In the study, a novel method for the construction of fragmented sheets of mesenchymal stem cells (MSCs) with a uniform size, without treating with any enzymes, was reported. The obtained MSC sheets preserved the intercellular junctions and endogenous extracellular matrix and kept their cell phenotype. After injection through a needle, the fragmented MSC sheets maintained intact and retained their activity upon transferring to another growth surface, while the complete cell sheets were torn into pieces. Transplantation of fragmented MSC sheets in the skeletal muscle of a syngeneic rat model via local injection was evaluated. The transplanted MSC sheets were mainly localized at the site of injection, while the dissociated MSCs were scattered around. Additionally, there were significantly more MSCs retained in the local skeletal muscle for the group injected with fragmented MSC sheets than that injected with dissociated MSCs. These results indicated that the fragmented cell sheets might be used as a novel therapeutic cell-carrier for intramuscular administration.