The α_vβ₃ integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to α_vβ₃, inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of α_vβ₃ integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC₅₀ = 0.1 nM) than those required to detach osteoclasts from bone (IC₅₀ ∼1 µM); (2) echistatin (IC₅₀ = 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) α_vβ₃ integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), α_vβ₃ disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC₅₀ = 13 nM), and α_vβ₃ visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that α_vβ₃ integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC₅₀ = 0.1 nM) and maintenance of the sealing zone (IC₅₀ ∼10 nM).