Matrix extracellular phosphoglycoprotein (MEPE) belongs to the SIBLING protein family which play key roles in biomineralization. Although the growth plates of MEPE-overexpressing mice display severe morphological disruption, the expression and function of MEPE in growth plate matrix mineralization remains largely undefined. Here we show MEPE and its cleavage product, the acidic serine aspartate-rich MEPE-associated motif (ASARM) peptide, to be localised to the hypertrophic zone of the growth plate. We also demonstrate that the phosphorylated (p)ASARM peptide inhibits ATDC5 chondrocyte matrix mineralization. Stable MEPE-overexpressing ATDC5 cells also had significantly reduced matrix mineralization in comparison to the control cells. Interestingly, we show that the addition of the non-phosphorylated (np)ASARM peptide promoted mineralization in the ATDC5 cells. The peptides and the overexpression of MEPE did not affect the differentiation of the ATDC5 cells. For a more physiologically relevant model, we utilized the metatarsal organ culture model. We show the pASARM peptide to inhibit mineralization at two stages of development, as shown by histological and μCT analysis. Like in the ATDC5 cells, the peptides did not affect the differentiation of the metatarsals indicating that the effects seen on mineralization are direct, as is additionally confirmed by no change in alkaline phosphatase activity or mRNA expression. In the metatarsal organ cultures, the pASARM peptide also reduced endothelial cell markers and vascular endothelial growth factor mRNA expression. Taken together these results show MEPE to be an important regulator of growth plate chondrocyte matrix mineralization through its cleavage to an ASARM peptide.