Around the world, sexual prejudice is still a concerning attitude present in health care providers and institutions. Identification of sexual prejudice during medical training could be an initial strategy to improve health care assistance to this particular population. Despite significant knowledge in the area, almost all previous studies were based on samples from developed countries, and there are no studies evaluating sexual prejudice and its conditionings among Latin American medical students. Objectives of the study were to estimate prevalence and predictors of sexual prejudice among medical students from two large cities in Colombia. This research found that 22.9% of medical students had a high sexual prejudice. Males and individuals with high religiosity were associated with a higher sexual prejudice and may require more sensitization and education in the area. Medical training represents a very good opportunity to approach the problem and decrease sexual prejudice in future physicians.