Numerous clinical conditions have been linked to ectopic mineralization (EM). This process of pathological biomineralization is complex and not fully elucidated, but thought to be started within matrix vesicles (MVs). We hypothesized that high mobility group box 1 (HMGB1), a cytokine associated with biomineralizing process under physiological and pathological conditions, induces EM via promoting MVs secretion from macrophages. In this study, we found that HMGB1 significantly promoted secretion of MVs from macrophages and subsequently led to mineral deposition in elevated Ca/Pi medium in vitro. Transmission electron microscopy of calcifying MVs showed formation of hydroxyapatite crystals in the vesicle interior. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase2 (nSMase2) that involved the receptor for advanced glycation end products (RAGE) and p38 MAPK (upstream of nSMase2). Inhibition of nSMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. Collectively, HMGB1 induces MVs secretion from macrophages at least in part, via the RAGE/p38 MAPK/nSMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 induces ectopic mineralization.