Osteoclasts are the primary mediators of pathological bone resorption in many conditions in which micro-environmental hypoxia is associated with disease progression. However, effects of hypoxia on human osteoclast activity have not been reported. Mature human osteoclasts were differentiated from peripheral blood or obtained from giant cell tumour of bone. Osteoclasts were exposed to a constant hypoxic environment and then assessed for parameters including resorption (toluidine blue staining of dentine slices), membrane integrity (trypan blue exclusion), apoptosis (TUNEL, DAPI), and osteolysis-associated enzyme activity (TRAP, cathepsin K). 24 h exposure to 2% O2 produced a 2.5-fold increase in resorption associated with increased TRAP and cathepsin K enzyme activity. Hypoxia-Inducible Factor-1α (HIF-1α) siRNA completely ablated the hypoxic increase in osteoclast resorption. 24 h at 2% O2 also increased the number of osteoclasts with compromised membrane integrity from 6% to 21%, with no change in the total osteoclast number or the proportion of late-stage apoptotic cells. Transient reoxygenation returned the percentage of trypan blue-positive cells to normoxic levels, suggesting that osteoclasts can recover from the early stages of cell death. Repeated over an extended period, hypoxia/reoxygenation enhanced osteoclast differentiation at this pO2. These data suggest that in diseased bone, where the pO2 may fall to ≤2% O2, a delicate balance between hypoxia-induced osteoclast activation and hypoxia-induced osteoclast apoptosis mediates pathological bone resorption. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.