Endoplasmic reticulum (ER) stress is well known as a suppressor in osteoblast differentiation and activating transcription factor 3 (ATF3) could be induced by a various extracellular signals including cytokines, hormones, DNA damage. Up to date, although the role of ATF3 have been studied, the function of ATF3 in osteoblast differentiation is still not clear yet. Our study showed that expression level of ATF3 could be incresed by tunicamycin which is ER stress inducer in preosteoblasts. BMPs, which are secreted by osteoblasts, can be important regulators in osteogenic differentiation. The stress-responsive transcription factor ATF3 is a negative regulator of osteoblast differentiation in MC3T3-E1 cells. In this study, we verified that BMP2-stimulated osteoblast differentiation could be inhibited by over-expressed ATF3 through regulating alkaline phosphatase (ALP) expression and activation.