Introduction: Teaching veterinary anatomy presents unique challenges due to the considerable morphological variation among domestic species, the limited availability of large specimen collections—especially for wild animals—and the biosafety restrictions associated with cadaveric materials. Three-dimensional (3D) printing has emerged as a promising tool to overcome these limitations by enabling the production of durable, safe, and anatomically accurate models. This study aimed to compare learning outcomes using three different study methods: real anatomical specimens, 3D-printed models, and traditional 2D materials (books and printed images). Methods: This study was conducted with undergraduate veterinary students (n = 261), divided into two subgroups based on prior exposure to anatomy: the 'previous knowledge group' (n = 111), composed of students who had already completed the anatomy course, and the 'no prior knowledge group' (n = 150), composed of first-semester students. Participants were randomly assigned to one of the three study methods and given one hour to study the assigned material. All students subsequently completed the same practical test using real specimens, allowing for direct comparison of performance across methods. Results: Students who studied using real anatomical specimens or 3D-printed models achieved significantly higher test scores compared to those who studied exclusively with 2D materials, with no statistical difference between the real-specimen and 3D-model groups. Test completion time did not differ among groups, and no significant differences were found between male and female participants. Conclusions: These findings demonstrate that 3D-printed models are a reliable tool for anatomical learning, providing outcomes equivalent to real specimens. They also offer practical advantages such as lower costs, improved biosafety, durability, and color customization. Moreover, 3D printing—and related technologies—holds strong potential for broader applications in veterinary and health sciences education, surgical planning, skills training, and more ethical and scalable research involving animals.