Background: Anatomy is one of the core subjects in medical education. Students spend considerable time and effort on learning the requisite anatomy knowledge. This study explored the effect of a multiple-player virtual reality (VR) gaming system on anatomy learning. Methods: 18 participants were randomly assigned into 3 learning conditions: (1) a textbook reading control group (CG), (2) a single-player VR (SP) group; and (3) a multiple-player VR (MP) group. The participants studied anatomy for 5 days, and completed a multiple-choice test on Days 1, 5, and 12. In the VR environment, the participants used handheld controllers to move the simulated tissues. The mission of the game was to complete puzzles of a human body. The SP and MP groups filled out a motivation inventory on Day 5. The scores on the multiple-choice test, the correct assembly rates, and the motivation inventory scores were analyzed using the 2-way ANOVA or independent t-test to compare group differences. Results: There was a significant interaction effect of group and timepoint (p = 0.003) in the multiple-choice test. In the CG, the scores on Day 1, Day 5, and Day 12 were significantly different (p < 0.001). The scores on Day 5 were significantly higher than those on Day 1 (p < 0.001). Although the scores declined slightly on Day 12, they were still significantly higher than those on Day 1 (p < 0.001). The SP and MP groups had similar results (p < 0.001, p < 0.001). The differences between the groups were only significant on Day 12 (p = 0.003), not Day 5 (p = 0.06). On Day 12, the scores of the MP group were higher than those of the CG (p = 0.002). The SP group and MP group had high scores on the interest, competence, and importance subscales of the motivation inventory. Both VR groups considered the system to be fun and beneficial to their learning. However, the MP group reported higher stress levels than the SP group. Conclusion: The results indicated that the proposed VR learning system had a positive impact on the anatomy learning. Although the between-player competition caused higher stress levels for the VR groups, the stress could have been a mediator of their learning outcomes. Trial registration: ETRD, ETRD-D-19-00573. Registered 20 December 2018, http://www.edah.org.tw/irb/index.htm.
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