Objective To review the research progress and clinical prospect of three-dimensional spheroid culture of mesenchymal stem cells (MSCs). Methods Recent literature about three-dimensional spheroid culture of MSCs was summarized, mainly on the formation of MSCs spheroids collected by three-dimensional culture, differences between MSCs spheroids and MSCs collected by traditional two-dimensional culture, and the mechanism underlying these differences. Last, its clinical prospect was discussed. Results Compared with MSCs collected by traditional two-dimensional culture, MSCs spheroids collected by three-dimensional culture get a salient up-regulation in anti-apoptosis, multiple differentiation potential, paracrine, and anti-inflammatory effect, which may be related to the morphology and cytoskeleton organization, cell-to-cell contact and gap junctions, and the hypoxia microenvironment. The animal experiments show obvious effects in repair of refractory wounds, repair of ischemic injury, and tissue remodeling, so MSCs spheroid has broad clinical prospect. Conclusion MSCs spheroids collected by three-dimensional culture have stronger biological potential and treatment effect than MSCs collected by traditional two-dimensional culture, MSCs spheroids can be used to optimize stem cell therapy and improve its treatment effect.