Ion transporters such as plasma membrane Ca 2+-ATPase (PMCA) and Na+/Ca2+ exchanger (NCX) are involved in the delivery of Ca2+ into mineralizing osteoid by osteoblast. However, little is known of the function of Na,K-ATPase in osteoblasts. This study was designed to elucidat e the role of Na,K-ATPase in osteoblast proliferation, differentiation and mineralization. We investigated Na, K-ATPase activity in developing osteoblastic MC3T3-E1 (E1) cells. Cell proliferation, alkaline phosphatase (ALP) activity and the deposition of mineral were assessed under the presence of a Na,K-ATPase specific inhibitor, ouabain. Effects of Na,K-ATPase inhibition on BMP-2- induced mineralization, expression of the osteoblast phenotype marker gene and the phosphorylation of Smad1/5/8 and MAPK in E1 cells were examined using ouabain and small interfering RNA. Na,K-ATPase activity was highest before confluence, tapered off quickly and then transiently increased at 18 days after confluence. When treated with ouabain around 18 days after confluence, mineralization fell, reflecting lower ALP activity in E1 cells. Likewise, inhibition of Na,K-ATPase reduced mineral deposition, and the mRNA expression of Runx2, ALP and osteocalcin. Pretreatment with ouabain was only weakly effective on BMP-2-induced Smad1/5/8 phosphorylation at Ser463 and Ser465 in the C-terminal region. On the other hand, ouabain induced the phosphorylation at Ser206 in the linker region on Smad1. Additionally, ouabain increased phosphorylation of ERK and p38 in response to BMP-2, whereas JNK phosphorylation was suppressed. The results of this study suggest Na,K-ATPase could regulate mineralization via MAPK as well as cell proliferation in osteoblasts.