Loss of mechanical loading induces rapid bone loss resulting from reduced osteoblastogenesis and decreased bone formation. The signaling mechanisms involved in this deleterious effect on skeletal metabolism remain poorly understood. We have previously shown that hindlimb suspension in rats increases osteoblast apoptosis associated with decreased phosphatidylinositol 3-kinase (PI3K) signaling. In this study, we investigated whether transforming growth factor (TGF)-beta2 may prevent the altered signaling and osteoblast apoptosis induced by skeletal unloading in vivo. Hindlimb suspension-induced decreased bone volume was associated with reduced alpha(5)beta(1)-integrin protein levels and PI3K/Akt signaling in unloaded bone. Continuous administration of TGF-beta2 using osmotic minipumps prevented the decreased alpha(5)beta(1)-integrin expression and the reduced PI3K/Akt signaling in unloaded bone, resulting in the prevention of osteoblast apoptosis. We also show that TGF-beta2 prevented the decreased Bcl-2 levels induced by unloading, which suggests that TGF-beta2 targets Bcl-2 via PI3K/Akt to prevent osteoblast apoptosis in unloaded bone. Furthermore, we show that TGF-beta2 prevented the decrease in phosphorylated Bad, the inactive form of the proapoptotic protein Bad, induced by unloading. These results identify a protective role for TGF-beta2 in osteoblast apoptosis induced by mechanical unloading via the alpha(5)beta(1)/PI3K/Akt signaling cascade and downstream Bcl-2 and phospho-Bad survival proteins. We thus propose a novel role for TGF-beta2 in protection from unloading-induced apoptosis in vivo.