To assess the role of microtubule acetylation in the transportation of amorphous calcium phosphate (ACP)-containing vesicles that mediate the osteogenic differentiation process of rat bone mesenchymal stem cells (BMSCs).Rat BMSCs were cultured and transfected with sirtuin 2 (SIRT2) overexpression plasmids for an in vitro model. The microtubule acetylation-related protein levels were detected by western blots. The microtubule acetylation and the secretion rate of extracellular ACPcontaining vesicles were observed with immunofluorescence and live cell fluorescence imaging. The secretion of ACP was observed by transmission electron microscopy. The mineralised nodule formation was stained with Alizarin Red S staining and observed by microscopy.Microtubule acetylation was increased during osteogenic differentiation of BMSCs, and microtubule transport efficiency was enhanced. Mechanically, microtubule acetylation is the key reason for the increased transportation rate of ACP-containing vesicles and enhanced osteogenic differentiation, as both were blocked after SIRT2-mediated microtubule acetylation inhibition.Microtubule acetylation mainly promotes the transportation and secretion of ACP vesicles, and ultimately promotes the osteogenic differentiation process.