ABSTRACT Background: This research assesses the level of knowledge and attitudes of medical students in South India on organ donation and transplantation, focusing on cultural and psychological barriers that hinder these practices, especially in developing regions. Methods: The cross-sectional research was carried out at SRM Medical College and Hospital and Research Centre in Chennai, Tamil Nadu, involving 207 undergraduate medical students. Data were collected through a structured questionnaire, evaluating knowledge and attitudes toward organ donation. The Knowledge Subscale contained 13 questions, while the Attitude Subscale had 13 items rated on a five-point Likert scale. Results: The study found that 57.59% of participants were female and aged 18–21, with 97.58% aware of organ donation and the “Transplantation of Human Organs Act.” However, only 7.73% knew where to obtain donation cards. Attitudes toward organ donation were mixed, with discomfort and religious beliefs being significant barriers. Concerns about body disfigurement and premature medical treatment persisted. Conclusion: The study emphasizes the need for educational interventions to improve medical students’ understanding and attitudes toward organ donation in South India, considering demographic, personal, and cultural factors, and suggests a multifaceted approach for community engagement.