Background: Human Papillomavirus (HPV) is the most common sexually transmitted infection that affects teens and adults in their early 20 s. Screening and HPV vaccination are important preventive measures to reduce cases of HPV and associated complications. Studies about HPV knowledge and vaccine acceptability are scarce in Saudi Arabia. This study evaluated participants’ knowledge, attitude, and acceptability concerning HPV and the HPV vaccine in Jazan Province, Saudi Arabia. Methods: A cross-sectional study was conducted using a pretested survey that assessed knowledge and attitude toward HPV and the HPV vaccine among the population of Jazan Province, Saudi Arabia. Responses were collected from adults between January and March 2022. Data were analyzed using a t-test and chi-square test. Results: We included 569 in the study. Most participants were single (65%), females (83%), with a university-level of education (78%). The recorded knowledge score for all participants was 1.99 (out of 10). About half of the participants denied that HPV is a common sexually transmitted infection. Further, 53% were interested in the HPV vaccine, and 63% of participants acknowledged that the HPV vaccine could prevent warts and cervical cancer. About 30% of the participants opposed the vaccine due to religious reasons. Conclusion: The results of our study highlight the inadequate level of knowledge concerning HPV infection, even among highly educated people. Thus, by promoting the HPV vaccine acceptance and use, awareness can be raised in our community to assure better knowledge and achieve higher protection from this virus and its complications.