Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE(2), a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE(2) by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels. The opening of hemichannels and release of PGE(2) are magnitude-dependent on the level of shear stress. Insertion of a rest period between stress enhances this response. Hemichannels gradually close after 24 h of continuous shear stress corresponding with reduced Cx43 expression on the cell surface, thereby reducing any potential negative effects of channels staying open for extended periods. These data suggest that Cx43-hemichannel activity associated with PGE(2) release is adaptively regulated by mechanical loading to provide an effective means of regulating levels of extracellular signaling molecules responsible for initiation of bone (re)modeling.