Osteocytes are believed to be the primary mechanosensors of bone tissue, signaling to osteoblasts and osteoclasts by releasing specific proteins. Sclerostin, interleukin-6 (IL-6), and insulin-like growth factor-I (IGF-I) are osteocyte proteins that signal to osteoblasts. The primary objective of this study was to determine if osteocyte protein response to mechanical unloading is restricted to the unloaded bone using the hindlimb unloading (HU) rodent model. We also examined tumor necrosis factor-α (TNF-α) due to its interactions with all three osteocyte proteins. We hypothesized that unloaded hindlimb cancellous bone would have an altered osteocyte protein (sclerostin, IL-6, and IGF-I) response compared to controls, while the response in the weight-bearing forelimb would not differ from ambulating controls. Male Sprague Dawley rats (7-mo old) experienced either HU (n=7) or normal cage activity (CON; n=7) for 28days. The unloaded distal femur and the weight-bearing proximal humerus were compared in HU vs CON. Metaphyseal bone density was reduced in the HU rats' hindlimb, but not in the proximal humerus, compared to CON values. Osteocyte density was 30% lower in the HU distal femur, but not different from CON in the proximal humerus. %Sclerostin+osteocytes in the distal femur were higher in HU compared to CON, but lower in the proximal humerus. Both %IGF-I+ and %IL-6+ osteocytes were lower in the distal femur for HU, but higher in the proximal humerus for HU. Osterix surface, a marker of osteoblasts, was lower in HU in the distal femur; however, the proximal humerus had more %osterix+surface in HU. In HU %Cathepsin K+ surface, a marker of osteoclasts, was higher in the distal femur and lower in the proximal humerus. %TNF-α+osteocytes were no different from CON in either bone site. HU proximal humerus osteocyte protein responses of sclerostin, IL-6, and IGF-I changed in the opposite direction as observed in the distal femur within the same animal. The opposite response of osteocyte proteins and osteoblast surface in hind- and forelimb bones within the same animal suggests that, while osteocytes in the unloaded hindlimb sense a lack of mechanical strain, osteocytes in the weight-bearing forelimb in HU animals sense some increase in local strain and generate molecular signaling to osteoblasts.