Exosomes, small extracellular vesicles that function as a key regulator of cell-to-cell communication, are emerging as a promising candidate for bone regeneration. Here, we aimed to investigate the effect of exosomes from pre-differentiated human alveolar bone-derived bone marrow mesenchymal stromal cells (AB-BMSCs) carrying specific microRNAs on bone regeneration. Exosomes secreted from AB-BMSCs pre-differentiated for 0 and 7 days were cocultured with BMSCs in vitro to investigate their effect on the differentiation of the BMSCs. MiRNAs from AB-BMSCs at different stages of osteogenic differentiation were analyzed. BMSCs seeded on poly-L-lactic acid(PLLA) scaffolds were treated with miRNA antagonist-decorated exosomes to verify their effect on new bone regeneration. Exosomes pre-differentiated for 7 days effectively promoted the differentiation of BMSCs. Bioinformatic analysis revealed that miRNAs within the exosomes were differentially expressed, including the upregulation of osteogenic miRNAs (miR-3182, miR-1468) and downregulation of anti-osteogenic miRNAs (miR-182-5p, miR-335-3p, miR-382-5p), causing activation of the PI3K/Akt signaling pathway. The treatment of BMSC-seeded scaffolds with anti-miR-182-5p decorated exosomes demonstrated enhanced osteogenic differentiation and efficient formation of new bone. In conclusion, Osteogenic exosomes secreted from pre-differentiated AB-BMSCs were identified and the gene modification of exosomes provides great potential as a bone regeneration strategy. DATA AVAILABILITY STATEMENT: Data generated or analyzed in this paper partly are available in the GEO public data repository(http://www.ncbi.nlm.nih.gov/geo).