Adherence to antiretroviral therapy (ART) remains a challenge for HIV-infected children. In this cross-sectional study, we used structured interview-administered questionnaires and medical records to measure adherence levels and factors associated with adherence and viral suppression. We included 195 South African children aged 2.1–12.9 on ART. Adherence levels ranged between 20.5% (pill count) and 89.1% (self-report). Boys were less adherent according to self-report, girls were less adherent according to pill count. Caregivers ensured medication was taken when the condition directly affected daily life. Well-functioning families and families with high SES provide a context supportive of adherence. Non-disclosure and difficulties administering medication negatively affected adherence and viral suppression. This study shows challenging levels of adherence impacting directly on viral suppression in a South African paediatric HIV program. Gender roles, non-disclosure and difficulty administering medication may undermine adherence and should be taken into account for clinical guidelines, policy design and inform strategies.