Cell-sheet technology has already constituted an important part in the regenerative medicine. Nonetheless, oligonucleotide delivery that has been widely performed on isolated stem cells to foster specific function is rarely conducted on the cell sheets. This study is designed with the two-fold aims of verifying the feasibility of non-viral oligonucleotide delivery for the cell sheets and confirming the osteogenesis enhancing effect of antimiR-138 on the cell sheets composed of bone marrow mesenchymal stem cells (BMSCs). The BMSC sheets are fabricated by a vitamin C inducing method, which can be successfully delivered with the oligonucleotides with a high delivery efficiency of nearly 100% by the properly adapted and optimized Lipofactamine2000 based formulation. The antimiR-138 delivery significantly enhances the in vitro osteogenic differentiation of BMSC sheets, indicated by the higher alkaline phosphatase (ALP) production, denser extracellular matrix mineralization and up-regulated osteogenesis related genes including runt-related transcription factor-2 (RUNX2), osterix, ALP, osteocalcin and bone morphogenetic protein-2 at both mRNA and protein levels, compared to controls. Regarding the underlying mechanism, the antimiR-138 delivery down-regulates the endogenous miR-138 levels in the BMSC sheets, consequently activates the extracellular signal regulated kinases 1/2 pathway and enhances the RUNX2 expression. The in vivo results indicate a robust enhancing effect of the antimiR-138 delivery on the bone regeneration ability of BMSC sheets. Massive bone with good vascularization is regenerated by the antimiR-138 delivered BMSC sheets, showing immense clinical significance for bone defect repair/regeneration applications. More importantly, the feasibility of non-viral oligonucleotide delivery system for the cell sheets as verified by our study shall hold a general significance for the cell sheets of various cell type and therapeutic purposes.