Each year millions of children in low- and middle-income countries (LMIC) fail to reach their developmental potential due to factors including poverty, malnutrition, poor stimulation and HIV. Although vertically-acquired HIV can now be prevented, little is known about the impact of HIV exposure in fetal and early life on the development of the many HIV-negative children. The Siyakhula Cohort was established within the Canadian Grand Challenges ‘Saving Brains’ initiative, to support re-enrolment of strategic cohorts in LMIC. This unique cohort in rural South Africa includes 1536 HIV-negative children born to HIV-infected (HIV-exposed) and HIV-uninfected (unexposed) mothers, enrolled from the Africa Health Research Institute (AHRI)–formerly the Africa Centre for Population Health (Africa Centre). The cohort includes data on HIV exposure in fetal and early life, and other early life factors (including breastfeeding) known to impact on later health outcomes. At birth, all children benefited from the early Prevention of Mother-to-Child-Transmission of HIV services in the district, and a subgroup were part of an additional early life breastfeeding intervention, the Vertical Transmission Study (VTS). This cohort pre-dated antiretroviral treatment (ART) roll-out, allowing for examination of outcomes associated with HIV exposure without ART exposure in utero and during breastfeeding. Current assessments at ages 7–11 years collected data on growth, health, cognition (including executive function), education and emotional-behavioural outcomes at primary school age.