Despite potential advantages of three-dimensional fluoroscopy-based navigation, there still remain a lot of controversies about the indications of this technology, especially whether it is worthy of being used in placement of pedicle screws in lumbar spine. However, according to the inconsistent conclusions reported in the literature and our experiences, the traditional method relying on anatomical landmarks and fluoroscopic views to guide lumbar pedicle screw insertion is unable to meet the requirement of precise screw placement. Based on our observation, screw malposition seems to occur concomitant with vertebral axial rotation which is a ubiquitous phenomenon. Threedimensional fluoroscopy-based navigation can provide the most valuable axial images in real-time, so it may be useful for placement of pedicle screws in lumbar spine. This study was intended to evaluate the effect of axial rotation of lumbar vertebrae on the accuracy of pedicle screw placement using the traditional method, as well as assess the value of three-dimensional fluoroscopy-based navigation in improving the accuracy. Sixteen lumbar simulation models at different degrees of axial rotation (0°, 5°, 10°, and 20°), with every four assigned the same degree, were equally divided into two groups (traditional method group and three-dimensional fluoroscopy-based navigation group). Random placement of pedicle screws was carried out, followed by CT scan postoperatively. Then the outer pedicle cortex contours were depicted from reconstructed sectional pedicle images using Photoshop. The accuracy of pedicle screw placement was evaluated by determining the interrelationship between screw trajectory and pedicle cortex (quality), and measuring the shortest distance from pedicle screw axis to outer cortex of the pedicle (quantity). Eighty pedicle screws were implanted, respectively, in each group. In traditional method group, statistical difference existed in the accuracy of pedicle screw placement at different axial rotational degrees (P \ 0.05). With degrees increasing, the accuracy declined. The accuracy of threedimensional fluoroscopy-based navigation group was higher than traditional method group in vertebrae with axial rotation (P \ 0.01). In qualitative evaluation, the accuracy of the two methods had statistical difference when the degree was 20°, and in quantitative evaluation, statistical difference existed in 5°, 10°, and 20°of vertebral axial rotation.