Background: Racial and ethnic minorities are disproportionately affected by human papillomavirus (HPV)-related cancer, many of which could have been prevented with vaccination. Yet, the initiation and completion rates of HPV vaccination remain low among these populations. Given the importance of social media platforms for health communication, we examined US-based HPV images on Twitter. We explored inconsistencies between the demographics represented in HPV images and the populations that experience the greatest burden of HPV-related disease. Objective: The objective of our study was to observe whether HPV images on Twitter reflect the actual burden of disease by select demographics and determine to what extent Twitter accounts utilized images that reflect the burden of disease in their health communication messages. Methods: We identified 456 image tweets about HPV that contained faces posted by US users between November 11, 2014 and August 8, 2016. We identified images containing at least one human face and utilized Face++ software to automatically extract the gender, age, and race of each face. We manually annotated the source accounts of these tweets into 3 types as follows: government (38⁄298, 12.8%), organizations (161⁄298, 54.0%), and individual (99⁄298, 33.2%) and topics (news, health, and other) to examine how images varied by message source. Results: Findings reflected the racial demographics of the US population but not the disease burden (795⁄1219, 65.22% white faces; 140⁄1219, 11.48% black faces; 71⁄1219, 5.82% Asian faces; and 213⁄1219, 17.47% racially ambiguous faces). Gender disparities were evident in the image faces; 71.70% (874⁄1219) represented female faces, whereas only 27.89% (340⁄1219) represented male faces. Among the 11-26 years age group recommended to receive HPV vaccine, HPV images contained more female-only faces (214⁄616, 34.3%) than males (37⁄616, 6.0%); the remainder of images included both male and female faces (365⁄616, 59.3%). Gender and racial disparities were present across different image sources. Faces from government sources were more likely to depict females (n=44) compared with males (n=16). Of male faces, 80% (12⁄15) of youth and 100% (1⁄1) of adults were white. News organization sources depicted high proportions of white faces (28⁄38, 97% of female youth and 12⁄12, 100% of adult males). Face++ identified fewer faces compared with manual annotation because of limitations with detecting multiple, small, or blurry faces. Nonetheless, Face++ achieved a high degree of accuracy with respect to gender, race, and age compared with manual annotation. Conclusions: This study reveals critical differences between the demographics reflected in HPV images and the actual burden of disease. Racial minorities are less likely to appear in HPV images despite higher rates of HPV incidence. Health communication efforts need to represent populations at risk better if we seek to reduce disparities in HPV infection.