Mesenchymal Stem Cells (MSCs) are pluripotent cells that can be differentiated as osteoblasts, adipocytes, myocytes or chondrocytes depending on the culture condition. However, MSCs are known to lose their differentiation potency after long-term culture. Development of a new cell culture method to maintain their stemness is required for successful application of MSCs. Here, we revealed that low-intensity pulsed ultrasound (LIPUS) stimulation was useful for maintaining the MSC stemness as LIPUS inhibited the loss of osteogenic differentiation potency of osteo-progenitor cells induced by serial subculture. LIPUS also increased the transcriptional and phosphorylation level of Nanog, a crucial stem cell marker gene in a MSC cell line. We also found that LIPUS induced the secretion of extracellular adenosine triphosphate (ATP) in MSC. The treatments of the conditioned medium from LIPUS-stimulated MSC and exogenous ATP promoted Nanog expression. Thus, LIPUS may maintain the long-term differentiation potency of MSCs and osteo-progenitor cells by induction in Nanog transcript level and phosphorylation.