Scanning acoustic microscopy (SAM) provides high-resolution mapping of acoustic impedance related to tissue stiffness. This study investigates changes in tissue acoustic impedance resulting from mechanical loading in trabecular bone cores cultured in 3-D bioreactor. Trabecular bone cores were extracted from bovine sternum (n = 15) and ulna metaphysis (n = 15). From each bone, the samples were divided in three groups. The basal control (BC) group was fixed post-extraction, the control (C) and loaded (L) groups were maintained as viable in a controlled culture-loading cell over three weeks. Samples of L group underwent a dynamic compressive strain, whereas C samples were left free from loading. After three weeks, L and C samples were embedded in polymethylmethacrylate and all samples were explored with a 200-MHz SAM. For each specimen, the acoustic impedance distribution was obtained over flat and polished section of bone blocks prepared parallel to the loading axis. Our results showed that in basal controls, the acoustic impedance varied with bone anatomical location and was 15% higher in weight-bearing ulna compared with nonweight-bearing sternum. The comparison between loaded and nonloaded groups showed that sternum-only exhibited significant change in acoustic impedance (L vs. C sternum: +9%). This result suggests that when the applied load is comparable with the stress naturally experienced by a weight-bearing bone (ulna), the tissue material properties (manifested by acoustic impedance) remained unchanged. In conclusion, SAM is a potentially relevant tool for the assessment of subtle changes in intrinsic microelastic properties of bone induced by adaptive remodeling process in response to mechanical loading.