Background For the investigation of the biomechanical properties of bone, various testing devices have been described. However, only a limited number have been developed to test the vertebral body of small animals. The aim of this study was to develop and validate a new bone testing device, which investigates the different biomechanical properties in small-animal vertebrae as a whole, three-dimensional unit, respecting its anatomical structure. Methods Thirty-five twelve-week-old female Sprague Dawley rats were utilized. Group 1 was composed of 17 rats with a normal bone metabolism without osteoporosis, while Group 2 consisted of 18 rats with manifest osteoporosis, 8 weeks after ovariectomy. The 5th lumbar vertebra of each animal was tested using the new bone testing device. This device has the ability to be adjusted to the slanted nature of each individual vertebral body and fix the vertebra in a natural position to allow for a non-dislocating axial force application. The device is designed to respect the anatomical three-dimensional shape of the vertebral body, thus avoiding the application of non-anatomic, non-physiological forces and thus preventing a distortion of the biomechanical testing results. The parameters investigated were stiffness, yield load, maximum load and failure load, and the results were compared to current literature values. Results The conduction of the biomechanical bone testing of the vertebral bodies with the new device was conductible without any instances of dislocation of the vertebrae or machine malfunctions. Significant differences were found for stiffness, maximum load and failure load between groups, with a lower value in the osteoporotic rats in each parameter tested. The yield load was also lower in the osteoporotic group, however not significantly. The values achieved correlate with those in current literature. Conclusions This study demonstrates that the newly developed testing machine is easy to handle and produces valid data sets for testing biomechanical bone parameters of whole vertebral bodies in an established small animal model. Therefore, it can be utilized, also as reference data, to test different structural properties and changes in vertebral bone, for example, in different metabolic settings or under the influence of different pharmaceutical entities in further studies.