We assessed human papillomavirus (HPV) prevention practices and HPV literacy of pediatricians, family physicians and medical students in North Carolina. An online survey was distributed to physicians in 2018, and paper surveys were collected among medical students in 2019. Surveys measured HPV literacy, HPV prevention practices and HPV prevention self-efficacy. In terms of comfort, 27% of medical students and 24% of physicians anticipated having an uncomfortable conversion when recommending the vaccine to patients. Most physicians (76%, n = 230) followed the HPV vaccine age recommendation guidelines; however, those with higher HPV vaccine knowledge were more compliant with the guidelines (P < 0.01). Female physicians were more likely to start routinely recommending the HPV vaccine to women (84%, n = 134 versus 72%, n = 92) and men (81%, n = 127 versus 71%, n = 84) between the ages of 9 and 12 years (P < 0.05). Only 27%, n = 73 of physicians and 18%, n = 19 of medical students followed/knew the 'provider-driven' HPV-recommended style. Female physicians were more likely to use this communication style (32%, n = 48 versus 20%, n = 23, P = 0.03). HPV prevention curriculum should be incorporated into medical programs. The gender-related practice patterns found indicate a need for training of male providers specifically. Quality improvement efforts are needed for all physicians to strengthen vaccine communication, recommendation practices and guideline adherence.