Osteoporosis has long been associated with weak bones but recent studies have shown that bone tissue mineral becomes more heterogeneous and the expression of mechanosensors are altered during estrogen deficiency in an animal model of osteoporosis. However, whether these changes occur as a primary response to estrogen deficiency is unknown. In this study we investigate whether matrix production and mineralisation by mechanically-stimulated osteoblasts are impaired as a direct consequence of estrogen depletion. Osteoblast-like MC3T3-E1 cells were cultured for 14 days with 10⁻⁸M of 17β-estradiol and subsequently cultured with osteogenic media only, or supplemented with estrogen or an estrogen antagonist (Fulvestrant, 10⁻⁷M). Physiological shear stress (1Pa) was applied using an orbital shaker (290rpm, 40min/day), which allows long-term culture and induces oscillatory flow on cells. Osteoblasts phenotype, extracellular matrix (ECM), mineralisation and mechanosensors were tracked by qRT-PCR (Runx2, Col1a1, Col1a2, Cox2, Bglap2, FN1), by biochemical assays (ALP activity, DNA and calcium content), by immunostaining (integrin α_v, BSP2, fibronectin) and by labelling with calcein the calcium. The results of this study demonstrate that after 7 days, estrogen depleted cells had less integrin α_v mechanosensors compared to those that received continuous estrogen treatment. By 14 days the ECM formation (calcium, fibronectin) by osteoblasts was altered under estrogen depletion, when compared to cells that were cultured continuously with estrogen. This study provides evidence of changes in osteoblast behaviour under estrogen depletion, which might explain the alteration in tissue mineral content and the decrease of integrins observed previously in ovariectomized rats in vivo.