Following a successful Human Papilloma Virus (HPV) vaccination pilot in 2013–2015 in Kitui county, Kenya introduced the HPV vaccine in October 2019 with a goal to immunize approximately 800,000 girls annually against HPV. Our study assessed the knowledge, attitudes, and practice of affected groups towards HPV infection and vaccination in two counties of Kenya. Semi-structured interviews from children aged between nine and thirteen years and key informants comprising of parents, head teachers, community leaders and health workers involved in HPV vaccination in health facilities from Mombasa and Tana-River counties were conducted. Content was analyzed thematically and coded for emerging themes using the QRS Nvivo 12 Plus (QRS International, Doncaster, Australia) software package. From our findings, a significant proportion of participants, especially children, have limited knowledge of the subject. Vaccination of boys was opposed by most participants. Parents and the community members are not in favor of HPV vaccination, as compared to the other groups. A similar pattern of inadequate knowledge and strongly opposed attitudes was observed in Tana-River and Mombasa. Active community involvement in primary prevention strategies may promote the uptake of the vaccine which can be achieved by robust awareness, modifying the negative beliefs about HPV vaccine and encouraging the perceptibility of HPV vaccination.