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Background: Supplementation in lactating HIV-1-infected women with preformed vitamin A and [beta]-carotene (VA/BC) increases the risk of mother-to-child transmission of HIV through breastfeeding. Identifying a biological mechanism to explain this unexpected finding would lend support to a causal effect. Objective: The aim of the study was to evaluate the effect of VA/BC or multivitamin (B complex, vitamin C, and vitamin E) supplementation of HIV-infected women on HIV shedding in breast milk during the first 2 y postpartum. Design: We quantified viral (cell-free) and proviral (cell-associated) HIV loads in breast-milk samples collected [less than or equal to] 15 d after delivery and every 3 mo thereafter from 594 Tanzanian HIV-1-infected women who participated in a randomized trial. Women received 1 of the following 4 daily oral regimens in a 2 x 2 factorial fashion during pregnancy and throughout the first 2 y postpartum: multivitamin, VA/BC, multivitamin including VA/BC, or placebo. Results: The proportion of breast-milk samples with detectable viral load was significantly higher in women who received VA/BC (51.3%) than in women who were not assigned to VA/BC (44.8%; P = 0.02). The effect was apparent [greater than or equal to]6 mo postpartum (relative risk: 1.34; 95% CI: 1.04, 1.73). No associations with proviral load were observed. The multivitamin bad no effects. In observational analyses, [beta]-carotene but not retinol breast-milk concentrations were significantly associated with an increased viral load in milk. Conclusions: VA/BC supplementation in lactating women increases the HIV load in breast milk. This finding contributes to explaining the adverse effect of VA/BC on mother-to-child transmission. [beta]-Carotene appears to have an effect on breast-milk viral load, independent of preformed vitamin A. This trial was registered at clinicaltrials.gov as NCT00197756. Am J Clin Nutr 2010;92:881-6. doi: 10.3945/ajcn.2010.29339.