Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP^−/− OPN^+/+) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 ± 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM α-actin and SM22α. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP^−/− OPN^+/+ at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.2 wk) than MGP^−/− OPN^+/+ counterparts (6.6 ± 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo.