Skin wound healing is a complex physiological process that maintains the integrity of the skin tissues, involving a variety of distinct cell types and signaling molecules. The specific signaling pathways or extracellular cues that govern the healing processes remain elusive. Microvesicles (MVs) have recently emerged as critical mediators of cell communication by delivery of genetic materials to target cells. In this study, we found the direct delivery of HEKa-MVs expressing miR-21 mimics significantly promoted the healing of skin wound in diabetic rats. In-depth studies showed that MV miR-21 promoted fibroblast migration, differentiation, and contraction, induced a proangiogenic process of endothelial cells and mediated a pro-inflammatory response. Mechanically, MV miR-21 might target specific essential effector mRNA in fibroblasts such as MMP-1, MMP-3, TIMP3, and TIMP4 to increase MMPs expression and enzymatic activities. Moreover, MV miR-21 regulated alpha-SMA and N-cadherin to induce fibroblast-myofibroblast differentiation. MV miR-21 up-regulated the IL-6 and IL-8 expressions and their secretion to amplify the immune response. Furthermore, MV miR-21 down-regulated PTEN and RECK in protein level, and activate MAPK/ERK signaling cascade, thereby promoting fibroblast functions. Thus, our study has provided for the first time the basis for the potential application of HEKa-MVs, and MV miR-21 in particular for wound healing.