Aim To investigate in vitro calcification of aortic medial cells and the acceleration by 25-hydroxycholesterol. Methods Aortic medial cells were obtained by explantation. Von Kossa staining was performed to show calcification. Insoluble calcium of cellular layer was determined by biochemical method. And osteocalcin in the media was analyzed with radioimmunoassay. Results Primary cells appeared two different types: cells with the features of parallel growth and negative von Kossa staining, and cells forming nodules with positive von Kossa staining. Passaged cells formed no nodules after 28 days. Cells treated with 25-hydroxycholesterol appeared many cell nodules with positive von Kossa staining. Both insoluble calcium and medium osteocalcin increased significantly. Conclusions There are two subgroups of the cultured aortic medial cells: one with the characters of smooth muscle cells; the other like micro vascular pericytes, which can calcify the extracellular matrix. 25-hydroxycholesterol can promote in vitro calcification. Osteocalcin secretion increases during pericyte-like cell calcification, suggesting that osteocalcin may take part in aortic calcification.