This study sought to derive an enhanced understanding of the complex intracellular interactions that drive bone loss in postmenopausal osteoporosis. We applied an in-vitro multicellular niche to recapitulate cell-cell signalling between osteocytes, osteoblasts and osteoclasts to investigate (1) how estrogen-deficient and mechanically loaded osteocytes regulate osteoclastogenesis and (2) whether ROCK-II inhibition affects these mechanobiological responses. We report that mechanically stimulated and estrogen-deficient osteocytes upregulated RANKL/OPG and M-CSF gene expression, when compared to those treated with 10 nM estradiol. Osteoclast precursors (RAW 264.7) cultured within this niche underwent significant reduction in osteoclastogenic gene expression (CTSK), and there was an increasing trend in the area covered by TRAP(+) osteoclasts (24% vs. 19.4%, p = 0.06). Most interestingly, upon treatment with the ROCK-II inhibitor, RANKL/OPG and M-CSF gene expression by estrogen-deficient osteocytes were downregulated. Yet, this inhibition of the pro-osteoclastogenic factors by osteocytes did not ultimately reduce the differentiation of osteoclast precursors. Indeed, TRAP and CTSK gene expressions in osteoclast precursors were upregulated, and there was an increased trend for osteoclast area (30.4% vs. 24%, p = 0.07), which may have been influenced by static osteoblasts (MC3T3-E1) that were included in the niche. We conclude that ROCK-II inhibition can attenuate bone loss driven by osteocytes during estrogen deficiency.