To investigate the relationship between icariin and the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and the signal pathway involved. We applied a universally accepted calcification model of VSMCs induced by β glycerophosphate. Then the VSMCs calcification was observed by treatment with icariin and/or inhibitors of estrogen receptors (ERs) and p38-mitogen-activated protein kinase (MAPK) signaling. Icariin inhibited osteoblastic differentiation and mineralization of VSMCs due to decreased ALP activity and Runx2 expression. Further study demonstrated that icariin exerted this suppression effect through activating p38-MAPK but not extracellular-regulated kinase, JNK or Akt. An inhibitor of p38-MAPK partially reversed the inhibitory effects of icariin on osteoblastic differentiation. Interestingly, treatment of VSMCs with an ER antagonist ICI182780 and a selective ERα receptor antagonist PPT attenuated icariin-mediated inhibition effect of VSMCs calcification, associated with suppression of p38-MAPK phosphorylation. Icariin inhibited the osteoblastic differentiation of VSMCs, and that the inhibitory effects were mediated by p38-MAPK pathways through ERα.