Osteocytes are mechanosensory cells which are embedded in calcified collagenous matrix. The specific native matrix of osteocytes affects their regulatory activity, i.e., transmission of signaling molecules to osteoclasts and/or osteoblasts, in the mechanical adaptation of bone. Unfortunately, no existing in vitro model of cortical bone is currently available to study the mechanosensory function of human osteocytes in their native matrix. Therefore, we aimed to develop an in vitro three-dimensional mechanical loading model of human osteocytes in their native matrix. Human cortical bone explants containing osteocytes in their three-dimensional native matrix were cultured and mechanically loaded by three-point bending using a custom-made loading apparatus generating sinusoidal displacement. Osteocyte viability and sclerostin expression were measured 1–2 days before 5 min loading and 1 day after loading. Bone microdamage was visualized and quantified by micro-CT analysis and histology using BaSO4 staining. A linear relationship was found between loading magnitude (2302–13,811 µɛ) and force (1.6–4.9 N) exerted on the bone explants. At 24 h post-loading, osteocyte viability was not affected by 1600 µɛ loading. Sclerostin expression and bone microdamage were unaffected by loading up to 8000 µɛ. In conclusion, we developed an in vitro 3D mechanical loading model to study mechanoresponsiveness of viable osteocytes residing in their native matrix. This model is suitable to study the effect of changed bone matrix composition in metabolic bone disease on osteocyte mechanoresponsiveness.