A characteristic sign of zinc deficiency is retarded skeletal growth, but the role of zinc in osteoblasts is not well understood. Two major events for bone formation include osteoblast differentiation by bone marker gene expression, which is mainly regulated by bone-specific transcription factor Runx2 and extracellular matrix (ECM) mineralization by Ca deposits for bone nodule formation. We investigated whether zinc deficiency down-regulates bone marker gene transcription and whether this might occur through modulation of Runx2. We also investigated whether zinc deficiency decreases ECM mineralization in osteoblastic MC3T3-E1 cells. In the presence of 5 mumol/L TPEN as zinc chelator, zinc deficiency (ZnD: 1 micromol Zn/L) decreased bone marker gene (collagen type I, osteopontin, alkaline phosphatase, osteoclacin and parathyroid hormone receptor) expression, as compared to normal osteogenic medium (OSM) or zinc adequate medium (ZnA: 15 micromol/L) (P<0.05) both at 5 days (proliferation) and 15 days (matrix maturation). Decreased bone marker gene transcription by zinc deficiency could be caused by decreased nuclear Runx2 protein (P=0.05) and transcript (P<0.05) levels in ZnD. Furthermore, within the first 24 h of differentiation when Runx2 expression is induced, maximal Runx2 mRNA and nuclear protein levels were delayed in ZnD compared to OSM and ZnA. ECM Ca deposition was also lower in ZnD, which was also indirectly confirmed by detection of decreased cellular (synthesized) and medium (secreted) ALP activity as well as matrix ALP activity. Taken together, zinc deficiency attenuated osteogenic activity by decreasing bone marker gene transcription through reduced and delayed Runx2 expression and by decreasing ECM mineralization through inhibition of ALP activity in osteoblasts. Decreased and delayed bone marker gene, Runx2 expression and ECM mineralization in osteoblasts by zinc deficiency can be a potential explanation for the retarded skeletal growth which is the major zinc deficiency syndrome.