<p>Children living with HIV are at risk for iron deficiency, yet optimal strategies for prevention and treatment remain unclear. Here, we investigate iron absorption, losses, and the efficacy and safety of oral iron supplementation with versus without prebiotics in three prospective studies in children with virally suppressed HIV in South Africa (NCT03572010, NCT04931641). In the first study, using stable iron isotopes, we show that iron absorption from iron-fortified maize porridge, a lipid-based nutrient supplement, and an oral iron supplement is comparable between children with HIV (<i>n</i> = 43) and without HIV (<i>n</i> = 45). In the second study, we use a stable iron isotope dilution method over a 6-month period to demonstrate that children with HIV (<i>n</i> = 29) absorb significantly less iron from their habitual diet than their uninfected peers (<i>n</i> = 36), while basal iron losses are similar. In the third study, a 12-week randomised, placebo-controlled, double-blind trial, iron-deficient children with HIV receiving iron with prebiotic galacto-oligosaccharides (<i>n</i> = 41) exhibit a 39% greater relative increase in serum ferritin (primary outcome) compared to those receiving iron with placebo (<i>n</i> = 42) (<i>p</i> = 0.053). They also report significantly fewer infection-related symptoms, with no significant differences in gut inflammation or enteropathogen carriage (secondary outcomes). Collectively, these findings indicate that while dietary iron absorption is reduced in children with virally suppressed HIV, supplemental and fortificant iron are well absorbed, and co-administration of iron supplements with prebiotics may improve efficacy and safety.</p>