Recommendation by a healthcare provider is critical to increase human papillomavirus (HPV) vaccine uptake in the US. However, current deficits in providers' knowledge of HPV and its vaccine are not fully understood and interventions to amend knowledge gaps are untested. To determine whether attending a structured presentation could increase provider knowledge of the HPV vaccine, we assessed knowledge levels of physicians, non-physician healthcare workers, and medical students before and after attending a 30-minute lecture held between October 2012 and June 2014. Paired t-test and McNemar's test were used to compare knowledge scores and the proportion of correct responses for each question, respectively. Multiple linear regression analyses were performed to examine correlates of baseline knowledge and change in knowledge scores post-intervention. A total of 427 participants, including 75 physicians, 208 medical students, and 144 nurses or other healthcare workers, attended one of 16 presentations and responded to both pre-test and post-test surveys. Baseline knowledge was low among all groups, with scores higher among older participants and physicians/medical students. On average, knowledge scores significantly improved from 8 to 15 after the presentation (maximum possible score 16) (P < .001), irrespective of specialty, race/ethnicity, gender, and age. Although lower at baseline, knowledge scores of younger participants and non-physician healthcare workers (e.g., nurses, physician assistants (PAs), nursing students) improved the most of all groups. We conclude that a brief, structured presentation increased HPV knowledge among a variety of healthcare workers, even when their baseline knowledge was low.