Breastfeeding mothers with HIV infection not qualifying for antiretroviral therapy (ART) based on country-specific guidelines at the time of the Promoting Maternal-Infant Survival Everywhere trial and their uninfected neonates were randomized to maternal ART (mART) or infant nevirapine prophylaxis (iNVP) postpartum. HIV transmission proportions were similar (<1%) in the 2 arms. We assessed whether maternal viral load (MVL) and CD4 cell counts were associated with breastfeeding HIV transmission. MVL was collected at entry (7-14 days postpartum) and at weeks 6, 14, 26, and 50 postpartum. CD4 cell counts were collected at entry and weeks 14, 26, 38, and 50 postpartum. Infant HIV-1 nucleic acid test was performed at weeks 1 and 6, every 4 weeks until week 26, and then every 12 weeks. The associations of baseline and time-varying MVL and CD4 cell counts with transmission risk were assessed using time-to-event analyses by randomized treatment arm. Two thousand four hundred thirty-one mother-infant pairs were enrolled in the study. Baseline MVL (P = 0.11) and CD4 cell counts (P = 0.51) were not significantly associated with infant HIV-1 infection. Time-varying MVL was significantly associated with infant HIV-1 infection {hazard ratio 95% confidence interval (CI): 13.96 (3.12 to 62.45)} in the mART arm but not in the iNVP arm hazard ratio (95% CI): 1.04 (0.20 to 5.39). Time-varying CD4 cell counts were also significantly associated with infant HIV-1 infection hazard ratio (95% CI): 0.18 (0.03 to 0.93) in the mART arm but not in the iNVP arm hazard ratio (95% CI): 0.38 (0.08 to 1.77). In women receiving mART, increased MVL and decreased CD4 cell counts during breastfeeding were associated with increased risk of infant HIV-1 infection.