It has been proposed that estrogen regulates the mechanosensitivity of osteocytes; however, the effects of estrogen deficiency that arises during postmenopausal osteoporosis on mechanical stimulation induced calcium signaling in osteocytes remain elusive. Here, we pretreated MLO-Y4 osteocytes with 10 nM E2 for 2, 3 and 5 d, then simulated postmenopausal conditions either by estrogen withdrawal (EW) from culture medium, or by inhibiting the estrogen receptor by using fulvestrant and estrogen (FE; ICI 182,780) in vitro. We investigated [Ca2+](i) oscillations and mechanobiologic responses of osteocytes (EW and FE) that were exposed to oscillatory fluid flow (OFF; 1 Pa, 0.5 Hz). We demonstrated that estrogen treatment enhanced OFF-induced [Ca2+](i) oscillations and that this effect was abrogated both by FE and EW. Moreover, osteocytes in both estrogen-depleted groups (EW and FE) had reduced levels of NO and prostaglandin E2 release, down-regulated dentin matrix protein-1, sclerostin, osteopontin, osteocalcin, and alkaline phosphatase mRNA expression, and reduced F-actin fiber formation after OFF stimulation compared with estrogen-treated cells. We propose a link between estrogen deficiency and alterations in [Ca2+](i)-mediated mechanosensitivity of osteocytes, which ultimately alter osteocyte function and differentiation. Deepak, V., Kayastha, P., McNamara, L. M. Estrogen deficiency attenuates fluid flow induced [Ca2+](i) oscillations and mechanoresponsiveness of MLO-Y4 osteocytes.