Neuroanatomy is one of the most challenging subjects in anatomy, and novice students often experience difficulty grasping the complex three-dimensional (3D) spatial relationships. This study evaluated a 3D neuroanatomy e-learning module, as well as the relationship between spatial abilities and students' knowledge in neuroanatomy. The study's cross-over design divided the participants into two groups, each starting with tests for anatomy knowledge and spatial ability, followed by access to either the 3D online learning module or the gross anatomy laboratory. Participants completed a second knowledge test prior to accessing the other learning modality. Participants in both groups scored significantly higher on Quiz 1 than on the Pretest knowledge assessment (W = 47, P < 0.01; W = 30, P < 0.01). Students who initially accessed the 3D online resources scored significantly better on the Quiz 1 than students who accessed the gross anatomy resources (W = 397.5, P < 0.01). Scores significantly improved on Quiz 2 for participants who accessed the 3D learning module following exposure to the cadaveric resources (W = 94, P < 0.01). After exposure to both learning modalities, there were no significant differences between groups. Significant positive correlations were found between participants' spatial ability score and their performance on the Pretest, Quiz 1, and Quiz 2 assessments (r = 0.22, P = 0.04; r = 0.25, P = 0.02; r = 0.26, P = 0.02). These preliminary results found students appreciated working with the 3D e-learning module, and their learning outcomes significantly improved after accessing the resource. Anat Sci Educ 9: 431-439. © 2016 American Association of Anatomists.