Mesenchymal stem cells (MSCs) have drawn worldwide attention of scientists and clinicians due to their ability to differentiate into other cell lineages, secrete paracrine factors, modulate inflammation and immunity, and also due to the effectiveness of MSCs in treating degenerative diseases. Recent studies have shown that, when cultured in spheroids, MSCs have greater differentiation ability, and increased anti-inflammatory and immunomodulatory capacities compared with traditional two-dimensional (2D) cultures. Furthermore, spheroid MSCs can be used on scale-up productions in clinically relevant manufacturing platforms. Microvesicles (MVs) are small membranous vesicles that can transfer proteins, genetic materials, and lipids to cells. MVs derived from MSCs (MSC-MVs) are not only emerging as potent transfer agents for molecular information, but also are effective in a series of tissue repair and anti-tumor experiments. Therefore, both spheroid MSCs and MSC-MVs have great potential in experimental and clinical applications. In this review, the characteristics, therapeutic applications and potential clinical translational opportunities of spheroid MSCs and MSC-MVs were discussed.