Background <p>Macrophage-derived exosomes and exosomal microRNAs (miRNAs) are critical mediators of bone marrow mesenchymal stem cell (BMSC) differentiation. However, their regulatory mechanisms under bone morphogenetic protein 2 (BMP2) stimulation remain unclear.</p> Methods <p>This study identified macrophage-derived exosomes stimulated or unstimulated by BMP2 (BMP2-Exos and DMEM-Exos, respectively) and co-cultured them with BMSCs to assess the effects of BMP2-Exos on BMSC osteogenic differentiation (n = 3). Bioinformatic methods were used to analyze differentially expressed miRNAs, and RNA sequencing (RNA-Seq) was used to examine the exosomal miRNA expression profiles of BMP2-Exos and DMEM-Exos to elucidate underlying mechanisms.</p> Results <p>Six miRNAs were upregulated and one miRNA was downregulated after osteogenic induction. Gene ontology and pathway analyses revealed that the target genes of differentially expressed exosomal miRNAs regulate various biological processes, including protein binding, metabolic processes, and biological regulation, and are enriched in osteogenic differentiation-related pathways, such as the PI3K-Akt signaling pathway and regulation of the actin cytoskeleton. Bioinformatic analysis identified elevated expression of microRNA-466i-5p (miR-466i-5p) and microRNA-365-2-5p (miR-365-2-5p) in BMP2-Exos, which may promote the osteogenic differentiation of BMSCs.</p> Conclusions <p>BMP2-activated macrophage-derived exosomes regulate BMSC osteogenic differentiation through miR-466i-5p and miR-365-2-5p, thereby revealing a novel therapeutic target for exosome-based bone regeneration strategies.</p>