A trend in anatomical education is the development of alternative pedagogical approaches to replace or complement experiences in a cadaver laboratory; however, empirical evidence on their effectiveness is often not reported. This study aimed to evaluate the effectiveness of Anatomy Glove Learning System (AGLS), which enables students to learn the relationship between hand structure and function by drawing the structures onto a worn glove with imprinted bones. Massage therapy students (n = 73) were allocated into two groups and drew muscles onto either: (1) the glove using AGLS instructional videos (3D group); or (2) paper with palmar/dorsal views of hand bones during an instructor-guided activity (2D group). A self-confidence measure and knowledge test were completed before, immediately after, and one-week following the learning conditions. Self-confidence of hand anatomy in the 3D group gradually increased (3.2/10, 4.7/10, and 4.8/10), whereas self-confidence in the 2D group began to decline one-week later (3.2/10, 4.4/10, and 3.9/10). Knowledge of hand anatomy improved in both groups immediately after learning, (P < 0.001). Students' perceptions of AGLS were also assessed using a 10-pt Likert scale evaluation questionnaire (10 = high). Students perceived the AGLS videos (mean = 8.3 ± 2.0) and glove (mean = 8.1 ± 1.8) to be helpful in improving their understanding of hand anatomy and the majority of students preferred AGLS as a learning tool (mean = 8.6 ± 2.2). This study provides evidence demonstrating that AGLS and the traditional 2D learning approach are equally effective in promoting students' self-confidence and knowledge of hand anatomy.