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To evaluate the impact of different modalities of infant feeding on HIV transmission in children in a prevention of mother-to-child transmission (PMTCT) program in an urban hospital in Uganda.HIV-infected pregnant women in the PMTCT program at St Francis Hospital Nsambya, Kampala were offered the chance to participate in the study. Short-course antiretroviral regimens were provided and formula feeding offered free of charge for women choosing not to breastfeed. Mother–infant pairs were followed until 6 months postpartum. HIV status in children was assessed at week 6 and month 6. For the analyses, mother–infant pairs were classified into three groups according to the mode of infant feeding: exclusive formula feeding (EFF), exclusive breastfeeding (EBF) and mixed feeding (MF).A total of 306 children were enrolled. Transmission rates were 8.9% at week 6 (3.4% in the EFF group, 11.2% in the EBF group, 17.1% in the MF group) and 12.0% at month 6 (3.7% in the EFF group, 16.0% in the EBF group, and 20.4% in the MF group). The EBF and MF groups were associated with a significantly higher risk of HIV transmission than the EFF group. No significant risk difference was observed between the EBF and the MF groups.HIV transmission rates were significantly lower in formula-fed infants in comparison with both exclusively breastfed and mixed-fed infants. Transmission through breastfeeding seems to occur mainly in the first weeks after delivery.