Monoscopically projected three-dimensional (3D) visualization technology may have significant disadvantages for students with lower visual-spatial abilities despite its overall effectiveness in teaching anatomy. Previous research suggests that stereopsis may facilitate a better comprehension of anatomical knowledge. This study evaluated the educational effectiveness of stereoscopic augmented reality (AR) visualization and the modifying effect of visual-spatial abilities on learning. In a double-center randomized controlled trial, first- and second-year (bio)medical undergraduates studied lower limb anatomy with stereoscopic 3D AR model (n = 20), monoscopic 3D desktop model (n = 20), or two-dimensional (2D) anatomical atlas (n = 18). Visual-spatial abilities were tested with Mental Rotation Test (MRT), Paper Folding Test (PFT), and Mechanical Reasoning (MR) Test. Anatomical knowledge was assessed by the validated 30-item paper posttest. The overall posttest scores in the stereoscopic 3D AR group (47.8%) were similar to those in the monoscopic 3D desktop group (38.5%; P = 0.240) and the 2D anatomical atlas group (50.9%; P = 1.00). When stratified by visual-spatial abilities test scores, students with lower MRT scores achieved higher posttest scores in the stereoscopic 3D AR group (49.2%) as compared to the monoscopic 3D desktop group (33.4%; P = 0.015) and similar to the scores in the 2D group (46.4%; P = 0.99). Participants with higher MRT scores performed equally well in all conditions. It is instrumental to consider an aptitude-treatment interaction caused by visual-spatial abilities when designing research into 3D learning. Further research is needed to identify contributing features and the most effective way of introducing this technology into current educational programs.