3D visualization tools have been developed to assist in the anatomical learning of medical students, but the evidence on the effectiveness of these tools is inconsistent. Conventional 3D materials are often displayed on 2D screens, which can limit their educational impact. This study evaluates the effectiveness of the presentation of 3D learning materials using 3D displays compared to 2D displays in a randomized controlled trial. We developed glasses-free 3D learning materials for the anatomy of abdominal organs and blood vessels, which are compatible with the 3D spatial reality tabletop display EFL-SR1 developed by Sony. We assessed the effectiveness of these materials in a randomized controlled trial. Fifty-six medical students were randomly divided into two groups to learn using either 2D or 3D displays. The improvement in understanding of the spatial arrangement of anatomical structures was measured before and after the learning sessions using a novel 3D puzzle-like method. Both groups demonstrated improvements in understanding of the spatial arrangement of anatomical structures; there were no significant differences in overall performance between the 2D and 3D display groups. The 3D display group exhibited less variability in improvement indices, suggesting more consistent learning outcomes. The study indicates that glasses-free 3D displays can minimize learning disparities among medical students and provide a more universally effective learning environment for all students. The 3D learning materials compatible with glasses-free 3D displays and the innovative 3D puzzle-like method used in this study offer a novel approach to teaching and evaluating anatomy education.