Mesenchymal stem cells (MSCs) have been extensively applied in the field of tissue regeneration. MSCs derived from various tissues exhibit different characteristics. In this study, a cluster of cells were isolated from human ethmoid sinus mucosa membrane and termed as hESMSCs. hESMSCs was demonstrated to have MSC-specific characteristics of self-renewal and tri-lineage differentiation. In particular, hESMSCs displayed strong osteogenic differentiation potential, and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Next, hESMSCs were prepared into a cell sheet and combined with a PSeD scaffold seeded with rBMSCs to repair critical-sized calvarial defects in rats, which showed excellent reparative effects. Additionally, ELISA assays revealed that secreted cytokines, such as BMP-2, BMP-4 and bFGF, were higher in the hESMSCs conditioned medium, and immunohistochemistry validated that hESMSCs cell sheet promoted the expression of BMP signaling downstream genes in newly formed bone. In conclusion, hESMSCs were demonstrated to be a class of mesenchymal stem cells that possessed high self-renewal capacity along with strong osteogenic potential, and the cell sheet of hESMSCs could remarkably promote new bone regeneration, indicating that hESMSCs cell sheet could serve as a novel and promising alternative strategy in the management of bone regeneration.