Anatomy plays a key role in veterinary training, and alternatives to traditional teaching methods, such as game-based learning and escape rooms, are emerging as innovative and effective methods. However, the effectiveness of these approaches, particularly in areas such as veterinary anatomy, remains under-researched. The aim of this study was to evaluate the effectiveness of a digital escape room in teaching veterinary anatomy to first-year students at the Toulouse Veterinary School. It included interactive elements and puzzles based on veterinary anatomy content. Participants were randomly assigned to either an experimental group (n = 81) that engaged with the digital escape room or a control group (n = 81). Pre- and post-tests were conducted with a score out of 100 to assess knowledge acquisition, and the participants' perception of the digital escape room was evaluated using a questionnaire. When comparing the difference between pre- and post-test scores, the experimental group showed a statistically significant difference (p < 0.0001). When comparing the post-test, the experimental group achieved a higher test scores with statistical significance comparing to the control group (p < 0.0001), indicating improved knowledge acquisition. The questionnaire reported that the digital escape room helped increase motivation and interest in learning. However, the lowest score in student-teacher interaction indicates a potential lack of feedback to support student progress. Digital escape rooms are a promising effective and engaging tool for teaching veterinary anatomy, offering practicality, scalability, and potential application across various disciplines of veterinary studies.