Mesenchymal stem cells (MSCs) play an important role in regenerative medicine and drug discovery due to their multipotential differentiation capabilities and immunomodulatory effects. Compared with traditional 2D cultures of MSCs, 3D cultures of MSCs have emerged as an effective approach to enhance cell viability, proliferation, and functionality, and provide a more relevant physiological environment. Here, we review the therapeutic potential of 3D-cultured MSCs, highlighting their roles in tissue regeneration and repair and drug screening. We further summarize successful cases that apply 3D MSCs in modeling disease states, enabling the identification of novel therapeutic strategies. Despite these promising applications, we discuss challenges that remain in the clinical translation of 3D MSC technologies, including stability, cell heterogeneity, and regulatory issues. We conclude by addressing these obstacles and emphasizing the need for further research to fully exploit the potential of 3D MSCs in clinical practice.