We previously report that BMP2 mediates mild ER stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. The transcription factor activating transcription factor 6 (ATF6), a key regulator of the UPR, is known to be important for ER stress-mediated apoptosis and cell growth, but the molecular mechanism underlying these processes remains unexplored. In this study, we demonstrate that ATF6 is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. ATF6 can enhance the level of IRE1a-spliced XBP1S protein in chondrogenesis. IRE1a and ATF6 can synergistically regulate endogenous XBP1S gene expression in chondrogenesis. Furthermore, overexpression ATF6 inhibited, while ATF6-knockdown enhanced, the cell proliferation in chondrocyte development with G1 phase arresting, S phase reducing and G2-M phase delaying. Besides, Ad-ATF6 can activate, whereas knockdown ATF6 by an siRNA-silencing approach inhibited, ER stress-mediated apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, CHOP, p-JNK expression in the course of chondrocyte differentiation. On the other hand, FCM, TUNEL assay and immunohistochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that Ad-ATF6 activation of the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of ATF6 in regulating ER stress-mediated apoptosis in chondrocyte differentiation and the molecular mechanisms involved.