Lactoferrin (LF) is a breast milk glycoprotein with antimicrobial and anti-inflammatory effects. Its beneficial properties in infants, especially in those born preterm, are currently being studied in clinical trials. However, the maternal and nursing infant factors that may affect LF concentration in breast milk are still not clear. We conducted a systematic review to investigate the factors that may affect the concentration of LF in breast milk. We used a 2-step approach to identify the eligible studies according to inclusion/exclusion criteria, and to determine which studies would be considered. We included 70 qualified articles from 29 countries with publication dates ranging from 1976 to 2015. We described the correlation between LF concentration in breast milk and lactation stage; 10 maternal factors, such as race, parity, among others; and 2 infant factors: infections and prematurity. Colostrum has the highest LF levels, but they decrease with days postpartum. No other factor has been consistently associated with LF concentration. A major limitation of the majority of the published studies is the small sample size and the different methods used to measure LF concentration. Therefore, there is a need for large, multicenter studies with standardized study design, sample collection, and LF measurement methods to identify clinically significant factors associated with LF expression in breast milk, which will help promote exclusive breastfeeding in preterm infants.