Background: Our previous findings revealed that exosomes derived from M2-polarized macrophages enhance the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), and identified key microRNAs (miRNAs) using high-throughput miRNA sequencing. Therefore, the present study aimed to elucidate the role and underlying molecular mechanism by which exosomes derived from M2 macrophages mediate the osteogenic differentiation of hPDLSCs. Methods: Following lentiviral-mediated modulation of miR-6879-5p in both hPDLSCs and M2 macrophage-derived exosomes, RT-qPCR, western blotting, and Alizarin Red staining were applied to assess alterations in osteogenic markers, including ALP, OCN, Collagen I, and RUNX2, as well as mineralized nodule formation in hPDLSCs. Immunoprecipitation-mass spectrometry (IP-MS) was employed to identify proteins interacting with miR-6879-5p target genes in hPDLSCs. Results: Knockdown of miR-6879-5p in the exosomes reduced the expression of osteogenic markers and inhibited calcified nodule formation in hPDLSCs. Overexpression of TRIM26 attenuated the osteogenic differentiation of hPDLSCs, an effect that was reversed by miR-6879-5p overexpression. IP-MS identified 410 TRIM26-interacting proteins in hPDLSCs. These proteins were associated with ubiquitination, aerobic glycolysis, and amino acid metabolism. The hub proteins in the TRIM26-associated PPI network included RPL and RPS family proteins, as well as glycolysis-associated proteins. CO-IP confirmed an interaction between TRIM26 and PKM, and showed that TRIM26 increased PKM ubiquitination. Overexpression of PKM rescued TRIM26-mediated suppression of osteogenic marker expression and mineralized nodule formation in hPDLSCs. Conclusion: miR-6879-5p carried by M2 macrophage-derived exosomes promotes osteogenic differentiation and aerobic glycolysis in hPDLSCs via modulating TRIM26-mediated ubiquitination of PKM.