Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the main source of extracellular pyrophosphate. Along with tissue-nonspecific alkaline phosphatase (TNAP), ENPP1 plays an important role in balancing bone mineralisation. Although well established in pre-osteoblasts, the regulating mechanisms of ENPP1 in osteoblasts and osteocytes remain largely unknown. Using bioinformatic methods, osterix (Osx), an essential transcription factor in osteoblast differentiation and osteocyte function, was found to have five predicted binding sites on theENPP1promoter. ENPP1 and Osx showed a similar expression profile bothin vitroandin vivo. Over-expression of Osx in MC3T3-E1 and MLO-Y4 cells significantly up-regulated the expression of ENPP1 (p <0.05). The consensus Sp1 sequences, located in the proximalENPP1promoter, were identified as Osx-regulating sites using promoter truncation experiments and chromatin immunoprecipitation (ChIP) assays. The p38-mitogen-activated protein kinase (MAPK) signalling pathway was demonstrated to be responsible for ENPP1 promoter activation by Osx. Runt-related transcription factor 2 (Runx2) was confirmed to have synergistic effects with Osx in activatingENPP1promoter. Taken together, these results provided evidence of the regulating mechanisms ofENPP1transcription in osteoblasts and osteocytes.