Game-based learning can have a positive impact on medical education, and virtual worlds have great potential for supporting immersive online games. It is necessary to reinforce current medical students' knowledge about radiological anatomy and radiological signs. To meet this need, the objectives of this study were: to design a competition-based game in the virtual world, Second Life and to analyze the students' perceptions of Second Life and the game, as well as to analyze the medium-term retention of knowledge and the potential impact on the final grades. Ninety out of 197 (45.6%) third-year medical students voluntarily participated in an online game based on self-guided presentations and multiple-choice tests over six 6-day stages. Participants and non-participants were invited to perform an evaluation questionnaire about the experience and a post-exposure knowledge test. Participants rated the experience with mean scores equal to or higher than 8.1 on a 10-point scale, highlighting the professor (9.5 ± 1.1; mean ± SD) and the virtual environment (8.9 ± 1.1). Participants had better results in the post-exposure test than non-participants (59.0 ± 13.5 versus 45.3 ± 11.5; P < 0.001) and a lower percentage of answers left blank (6.7 ± 8.4 versus 13.1 ± 12.9; P = 0.014). Competitive game-based learning within Second Life is an effective and well-accepted means of teaching core radiological anatomy and radiological signs content to medical students. The higher medium-term outcomes obtained by participants may indicate effective learning with the game. Additionally, valuable positive perceptions about the game, the educational contents, and the potential benefit for their education were discovered among non-participants.