Recent in vivo studies have proposed that integrin alpha(v)beta(3) attachments between osteocyte cell processes and the extracellular matrix may facilitate mechanosensation in bone. However the role of these attachments in osteocyte biochemical response to mechanical stimulus has yet to be investigated. With this in mind, the objective of this study was to determine the effect of blocking integrin alpha(v)beta(3) function on the biochemical response of osteocytes to mechanical stimulus. Antagonists specific to integrin subunit beta(3) were used to block integrin alpha(v)beta(3) on MLO-Y4 mouse osteocytes. After treatment, cells were subjected to laminar oscillatory fluid flow stimulus (1 Pa, 1 Hz) for one hour. Fluorescent staining was performed to visualise cell morphology. Prostaglandin E-2 (PGE(2)) release was assayed using an enzyme immunoassay and qRT-PCR was used to analyse the relative expression of cyclooxygenase-2 (COX-2), receptor activator of NF-kappa B ligand (RANKL) and osteoprotegerin (OPG). Our results show that blocking integrin alpha(v)beta(3) disrupts osteocyte morphology, causing a reduction in spread area and process retraction. Integrin alpha(v)beta(3) blocking also disrupted COX-2 expression and PGE(2) release in response to fluid shear stress. Taken together, the results of this study indicate that integrin alpha(v)beta(3) is essential for the maintenance of osteocyte cell processes and also for mechanosensation and mechanotransduction by osteocytes. A better understanding of this process may lead to the development of novel treatments for bone pathologies where mechanosensitivity is thought to be compromised. (C) 2014 Elsevier Ltd. All rights reserved.