Objective To explores the role of medical museums in medical humanities education and clarifies their functional positioning, with the aim of developing a practice-oriented educational pathway. Methods Grounded in situated learning theory, a mixed-methods approach was employed. The museum complex of Shanghai Medical College, Fudan University served as the primary case, supplemented by a comparative analysis of publicly available information from medical museums worldwide. Subsequently, in June 2025, a questionnaire survey was administered to the museum′s docent team (n=55), and descriptive statistics were used to evaluate educational outcomes. Finally, an medical Humanities education pathway in medical museums was constructed by integrating theoretical analysis and empirical findings. Results As a situated learning environment, the medical museum effectively fosters the transformation of medical humanities education from knowledge acquisition to value internalization by constructing multi-dimensional situations, forming learning communities, and designing participatory activities. The survey showed that medical students deeply involved in museum activities showed the highest agreement on ″professional mission″ (4.67±0.47) and ″communication skills″ (4.67±0.47). Furthermore, a positive correlation was found between the depth of participation and educational outcomes. Conclusions The educational pathway developed in this study provides a theoretical framework and practical guidance for leveraging medical museums in medical humanities education, and can effectively enhance medical students′ humanistic literacy and professional identity.