Osteoporotic bone loss and fracture have long been regarded to arise upon depletion of circulating estrogen, which increases osteoclastogenesis and bone resorption. Osteoblasts from human osteoporotic patients also display deficient osteogenic responses to mechanical loading. However, while osteoblasts play an important role in regulating osteoclast differentiation, how this relationship is affected by estrogen deficiency is unknown. This study seeks to determine how mechanically stimulated osteoblasts regulate osteoclast differentiation and matrix degradation under estrogen deficiency. Here, we report that osteoblast-induced osteoclast differentiation (indicated by tartrate-resistant acid phosphatase, cathepsin K, and nuclear factor of activated T cells, cytoplasmic 1) and matrix degradation were inhibited by estrogen treatment and mechanical loading. However, estrogen-deficient osteoblasts exacerbated osteoclast formation and matrix degradation in conditioned medium and coculture experiments. This was accompanied by higher expression of cyclooxygenase-2 and macrophage colony-stimulating factor, but not osteoprotegerin, by osteoblasts under estrogen deficiency. Interestingly, this response was exacerbated under conditions that block the Rho-Rho-associated protein kinase signaling pathway. This study provides an important, but previously unrecognized, insight into bone loss in postmenopausal osteoporosis, whereby estrogen-deficient osteoblasts fail to produce inhibitory osteoprotegerin after mechanical stimulation but upregulate macrophage colony-stimulating factor and cyclooxygenase-2 expression and, thus, leave osteoclast activity unconstrained.