The P2X₇ nucleotide receptor (P2X₇R) is an ATP-gated ion channel expressed in many cell types including osteoblasts and osteocytes. Mice with a null mutation of P2X₇R have osteopenia in load bearing bones, suggesting that the P2X₇R may be involved in the skeletal response to mechanical loading. We found the skeletal sensitivity to mechanical loading was reduced by up to 73% in P2X₇R null (knock-out (KO)) mice. Release of ATP in the primary calvarial osteoblasts occurred within 1 min of onset of fluid shear stress (FSS). After 30 min of FSS, P2X₇R-mediated pore formation was observed in wild type (WT) cells but not in KO cells. FSS increased prostaglandin (PG) E2 release in WT cells but did not alter PGE₂ release in KO cells. Studies using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes confirmed that PGE₂ release was suppressed by P2X₇R blockade, whereas the P2X₇R agonist BzATP enhanced PGE₂ release. We conclude that ATP signaling through P2X₇R is necessary for mechanically induced release of prostaglandins by bone cells and subsequent osteogenesis.