The process of long bone growth involves the regulation of growth plate cartilage by vitamin D3 metabolites systemically and through local production by chondrocytes. In the post-fetal growth plate, chondrocytes align in columns and undergo maturation stages that are differentially regulated by 1α,25(OH)2D3 [1α,25] and 24R,25(OH)2D3 [24R,25]. Chondrocytes in the reserve zone (resting zone) generate a proteoglycan-rich extracellular matrix (ECM) and respond to 24R,25, stimulating their proliferation and regulating their production of ECM and matrix vesicles (MVs) containing neutral metalloproteinases (MMPs). 24R,25 upregulates their ability to respond to 1α,25, inhibiting proliferation and stimulating production of MVs containing acidic MMPs and matrix processing enzymes, nucleation sites for mineral formation, increased alkaline phosphatase activity, and secretion of Ca++ into the ECM. Vitamin D metabolites produced by chondrocytes act directly on MVs in the ECM. 24R,25 stabilizes the MV membranes while 1α,25 stimulates their breakdown, releasing MMPs. These vitamin D metabolites also modulate the incorporation of miRNA into the MVs and regulate their release to the ECM in a cell maturation-specific manner. When MVs are treated with 1α,25, miRNAs are not released, suggesting that they are protected by the membrane. 1α,25 also stimulates the uptake of MVs by mature chondrocytes, and miRNA present in the MVs are released into the cells. miRNA-122, which is found in the MVs produced by growth zone chondrocytes induces proliferation, stabilizes matrix formation, and reduces alkaline phosphatase in both resting zone and growth zone cells. This suggests that MVs act as a break on terminal chondrocyte differentiation. Chondrocyte proliferation and maturation in the female rat growth plate are regulated by 17β-estradiol, but the effects are modulated by 1α,25. In female growth zone cells, 1α,25 reduces the stimulatory effect of 17β-estradiol on alkaline phosphatase activity and increases the production of sulfated glycosaminoglycans, similar to the effects of MV miRNA on these cells. 1α,25 also stimulates osteoblast differentiation and MV production. miRNA-122 is enriched in MVs produced by osteoblasts and treatment of osteoblasts with MVs retards their terminal differentiation. These findings suggest that 1α,25 acts on growth zone chondrocytes and osteoblasts to promote endochondral bone formation, and provides a braking mechanism by packaging miRNA in MVs, stimulating their uptake by cells undergoing terminal differentiation.