Background Low national immunization coverage (44.64%) requires strengthening the vaccination campaign to improve knowledge about HPV and its vaccine among adolescents and parents/guardians. Our aim is to evaluate factors related to knowledge about HPV, its vaccine, acceptability and divergences among Brazilian adolescents and parents/guardians. Methods A cross-sectional study was performed at a health unit of Sao Paulo University, Brazil, from 2015 to 2016. The convenience sample comprised 1047 individuals, including 74% (n = 776) adolescents and 26% (n = 271) parents/guardians, who answered a survey (knowledge about HPV, its vaccine, barriers and acceptability). Results The main source of information for adolescents was school (39%, n = 298); for parents/guardians, it was health professionals (55%, n = 153). Parents/guardians were 2.48 times more likely than adolescents to know that HPV caused changes in the Pap smear test [RR 2.48, 95% CI 2.03–3.01 (p < 0.001)], 1.43 times likely to be aware that HPV was a sexually transmitted infection [RR 1.43, 95% CI 1.22–1.68 (p < 0.001)], and 2.77 times likely to be informed that the HPV vaccine decreased the chance of having genital warts [RR 2.77, 95% CI 2.22–2.47 (p < 0.001)]. Girls knew more about the topic than boys (RR 1.67; 95% CI 1.10–2.60); education increased parents’ knowledge [(RR 3.38; 95% CI 1.71–6.69)]. Conclusion Female adolescents and parents/guardians with a higher level of education are factors related to suitable knowledge about HPV and its vaccine among Brazilian respondents. There were differences between parents/guardians and adolescents in HPV awareness, clinical implications, vaccine knowledge and vaccine acceptance.