Background: Human papillomavirus (HPV) is a common sexually transmitted infection (STD) among young women, which is linked to a higher risk of cervical cancer. Cervical cancer incidence can be decreased by effective HPV vaccination and screening. Methods: A cross-sectional study was implemented using an Arabic language questionnaire that was administered electronically to parents of girl’s middle school students in AL Madinah AL Munawara from April to August 2023. The t-test and ANOVA test were used to analyze the collected data. Results: There were of 229 participants in this study; The findings showed that the level of parents’ knowledge about this virus was 42%; (37%) knew that HPV is an STD, 64% knew that HPV and cervical cancer are related, 63% were against their daughter getting the HPV vaccination. Furthermore, two-thirds of participants were concerned about the effectiveness of this vaccine, and 57% of them refused HPV vaccination due to moral or religious beliefs. The findings indicated also that parents' knowledge and acceptability of the HPV vaccine varied, with mothers being more knowledgeable than dads but fathers being more accepting of it. Conclusion: The results indicate insufficient knowledge about HPV infection, negative attitudes toward and limited acceptance of the HPV vaccination among parents of girls attending middle schools.