Obesity has reached epidemic levels, and physical education teachers are on the front lines to combat it. However, anti-fat bias from physical education teachers may be a barrier against the participation of students who are obese in physical activity. Thus, our purpose was to investigate the attitude of physical education teachers and majors toward obese individuals. Forty-seven physical education teachers and 149 majors participated in the study. Participants answered three questionnaires: Anti-Fat Attitude Scale (AFAS measures explicit attitudes toward obese individuals), Perception of Obese Students by Physical Education Teachers questionnaire (POSPET measures how teachers perceive students who are obese during class), and Implicit Association Test (IAT is a timed assessment measuring automatic attitudes toward obese individuals through word categorizations: good–bad, lazy–motivated). Based on one sample t tests, AFAS results indicated a neutral attitude by teachers (t46 = -1.63, p = .11) and majors (t148 = .80, p = .43) toward obese individuals, and POSPET results indicated teachers have a pro-fat bias toward obese students (t42 = -8.99, p < .01). However, when answers were automatically evoked, good–bad and lazy–motivated IAT scores indicated a strong anti-fat bias by teachers (t37 = 12.31, p < .01 and t35 = 13.12, p < .01, respectively) and majors (t137 = 16.96, p < .01 and t134 = 20.77, p < .01, respectively). Using independent t tests, significant differences between teachers and majors were not found for good–bad (t174 = .34, p = .74) and lazy–motivated IAT (t169 = -1.05, p = .21). IAT scores showed a strong and similar anti-fat bias among physical education teachers and majors. Anti-fat bias should be an important part of PETE and continuing education programs because it could play a role in the prevention of obesity.