BACKGROUND Antiretroviral (ARV) interventions are used to reduce HIV viral replication and prevent mother-to-child transmission. Viral suppression relies on adherence to ARVs. METHODS A 2-phase study was conducted using data from the Breastfeeding, Antiretrovirals, and Nutrition study. We included mothers randomized to 28 weeks of postpartum ARVs with ≥1 plasma or breastmilk specimen. All mothers who transmitted HIV to their infants from 2-28 weeks (n = 31) and 15% of mothers who did not (n = 232) were included. Adherence was measured by pill count [categorized as poor (0%-80%), partial (81%-98%), and near perfect (>98%)]. Associations between adherence and breastmilk RNA were assessed using mixed-effects models. Cox models were used to estimate associations between breastmilk RNA and HIV transmission. Using Monte Carlo simulation, we estimated the number of transmissions that would occur had everyone randomized to maternal ARVs been 90% and 100% adherent. RESULTS Partial or near perfect ARV adherence significantly reduced the odds of having detectable (≥40 copies/mL) breastmilk RNA, compared with poor adherence (Odds Ratio (OR) 0.23, 95% CI: 0.08 to 0.67; OR 0.36, 95% CI: 0.16 to 0.81, respectively). Detectable breastmilk RNA was associated with increased breastmilk transmission compared with undetectable breastmilk RNA (hazard ratio 3.8, 95% CI: 1.2 to 12.1). All transmitting mothers had ≥1 plasma viral load specimen >100 copies per milliliter. An estimated similar number of transmissions would occur with 90% adherence compared with 100%. CONCLUSIONS Helping patients adhere to ARVs throughout breastfeeding is important for realizing the full potential of recommended ARV interventions to prevent mother-to-child HIV transmission. Maintaining plasma viral load