Anti-fat bias by professors in physical education departments may interfere with the training provided to pre-service teachers. The purpose of this study was to evaluate the attitudes of professors in physical education departments toward obese individuals. Professors from randomly selected institutions across all four US regions participated in the study (N = 94). Participants took the Implicit Association Test and answered the Anti-Fat Attitude Scale and two questions specifically designed to measure their attitudes toward physical education teachers and majors who are obese. The participants exhibited implicit good-bad (p < 0.001) and lazy-motivated (p < 0.001) anti-fat biases. Professors favored accepting majors who are obese (p < 0.001), but they strongly disapproved of obese physical education teachers as role models to their students (p < 0.001). Explicit anti-fat bias was associated with a stronger disapproval of physical education teachers who are obese as role models to students (p < 0.001) and accepting majors who are obese (p < 0.001). Implicit good-bad anti-fat bias was associated with a stronger disapproval of obese physical education teachers as role models to students (p = 0.047). The anti-fat bias demonstrated by the professors may negatively affect the training of pre-service physical education teachers to work with students of all body sizes. Awareness programs may be necessary to diminish anti-fat bias among professors in physical education teacher education programs.