Bone sustains microfractures physiologically due to fatigue and supraphysiologically due to overload episodes. Bone cells target such damage for repair and actively modify the structure bone to better adapt to the mechanical environment. The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. A recently developed experimental model in our laboratory introduces notches (i.e. stress concentrator s) to bone slices to amplify mechanical stress controllably in a site-specific fashion which allows for realtime observation of the evolving damage in vitro. Upon the emergence of a damage zone in this model, we observe a partially reversible mechanically-induced exposure of nanostructural features of the same length scale with mineral crystals of bone and of positive charges similar to the calcium moieties in bone during post-yield deformation. By using an ultra-sensitive calcium microelectrode system, we further demonstrate a spontaneous efflux of calcium ions (Ca2+) (1.924 ± 0.742 pmol cm−2 s−1) originating from regions of devitalized bone matrix undergoing damage which are likely associated with calcium groups. When these notched bone sliced are seeded with MC3T3-E1 osteoblasts, the strain-induced Ca2+ efflux from bone elicits cell response at the stress concentration site as manifested by activation of intracellular calcium signaling (increase in fluorescence by 52% ± 27%). This activity is associated with extracellular calcium because the intracellular calcium signaling in response to mechanical loading subsides when experiments are repeated using demineralized bone substrates (increase in fluorescence by 6% ± 10%). Lastly, a wide range of pharmaceutical inhibitors were applied to identify the calcium entry pathways involved in the intracellular calcium signaling in response to the strain-induced calcium efflux from bone matrix: internal calcium release from endoplasmic reticulum (ER, inhibited by Thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by Calhex), stretch-activated calcium channel (SACC, inhibited by Gadolinium), voltage-gated calcium channels (VGCC, inhibited by Nifedipine, Verapamil, Neomycin, and ω-conotoxin), and calcium-induced-calciumrelease channel (CICRC, inhibited by Ryanodine and Dantrolene). The results showed only neomycin reduced the intracellular calcium response by about 75% for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. It suggests the intracellular calcium signaling occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. These results imply a novel perspective where bone matrix acts as an intermediary mechanochemical transducer by converting mechanical strain into a chemical signal (pericellular calcium) to which cells respond via VGCCs. Such a mechanism may be responsible for triggering repair at locations of bone matrix undergoing critical deformation levels which is essential to bone adaptation.