This study aimed to clarify how masticatory muscle atrophy induced by botulinum toxin (BTX) injection affects cortical bone quality of the mandible using 3D modeling technology. A total of 39 young (26.9 ± 6.0 years) and 38 post-menopausal (55.3 ± 6.3 years) females were included. Computed tomography (CT) images were obtained before and after 12 months of treatment. Predictor variables were application of a stabilization splint, and/or two times of BTX injection in the bilateral temporalis and masseter muscles within a six-month interval. Outcome variables were changes in average Hounsfield units (HU) and cortical thickness of region of interest (ROI). 3D mandibular models were reconstructed using CT images, and models were used to calculate average HU and cortical thickness of ROIs, including inferior half of the lateral surface of ascending ramus, coronoid process, and temporomandibular joint condyle. Cortical bone quality at muscle insertion site was influenced by decreased muscle thickness but seemed not to be affected by decreased functional loading. Reduced functional loading seemed to influence cortical bone quality of the condyles. These effects were more remarkable in post-menopausal females. Hence, decreased masticatory muscle thickness may lead to alterations of the mandibular cortical structures, especially in post-menopausal females.