The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca(2+) channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K-Akt signaling. Panx3 also formed gap junctions and propagated Ca(2+) waves between cells. Blocking the Panx3 Ca(2+) channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca(2+) channel and a hemichannel, and by forming gap junctions.