The integrin alpha(v)beta(3) has been shown to play an important role in osteocyte mechanotransduction. It has been reported that there are fewer beta(3) integrin-containing cells in osteoporotic bone cells. Osteocytes cultured in vitro under estrogen deficient conditions demonstrate altered mechanotransduction. However, it is unknown whether the altered mechanotransduction in estrogen deficient osteocytes is directly associated with defective alpha(v)beta(3) expression or signalling. The objective of this study is to investigate the role of estrogen deficiency for regulating MLO-Y4 cell morphology, alpha(v)beta(3) expression, focal adhesion formation and mechanotransduction by osteocytes. Here, we report that estrogen withdrawal leads to a smaller focal adhesion area and reduced alpha(v)beta(3) localisation at focal adhesion sites, resulting in an increased Rankl/Opg ratio and defective Cox-2 responses to oscillatory fluid flow. Interestingly, alpha(v)beta(3) antagonism had a similar effect on focal adhesion assembly, Rankl/Opg ratio, and Cox-2 responses to oscillatory fluid flow. Taken together, our results provide the first evidence for a relationship between estrogen withdrawal and defective alpha(v)beta(3)-mediated signalling. Specifically, this study implicates estrogen withdrawal as a putative mechanism responsible for altered alpha(v)beta(3) expression and resultant changes in downstream signalling in osteocytes during post-menopausal osteoporosis, which might provide an important, but previously unidentified, contribution to the bone loss cascade.