To investigate the knowledge and attitudes of medical graduate students regarding laboratory animal welfare, a survey was conducted on 288 medical graduate students from the 2019 to 2023 cohorts through the final exam of the compulsory course ‘Medical Laboratory Animal Science.’ The survey included both closed and open-ended questions. We calculated the awareness rate from the cumulative student score/maximum score based on the questions and scoring rubrics. We found that medical graduate students showed relatively low awareness of laboratory animal welfare and limited understanding of related knowledge. There were shortcomings in theoretical knowledge and practical experience related to laboratory animals’ welfare and ethical aspects of laboratory animal care and use. Students in basic medical disciplines demonstrated slightly better awareness of laboratory animal welfare than those in other disciplines. In addition, female students exhibited a higher awareness of laboratory animal welfare than male students. In recent years, there has been a gradual increase in the awareness of the importance of laboratory animal welfare among medical graduate students, but a decrease in awareness was observed during the COVID-19 pandemic. Therefore, a need exists for strengthening education on laboratory animal welfare in medical schools to enhance students’ awareness. It is suggested that school-specific expectations with respect to the replacement, reduction, and refinement principles be established to balance the needs of medical research and laboratory animal welfare, as a way to ensure the ethical considerations and the smooth progress of medical research are both met.