Summary Bioactive components of human milk, such as human lactoferrin (hLF), play an essential role in gut microbiome homeostasis and protection against neonatal inflammatory diseases. Neonatal intestinal macrophages display a proinflammatory profile that might contribute to inflammatory mucosal injury. Therefore, the aim of the study was to investigate the immunomodulatory effects of hLF on differentiation and activation of monocyte-derived macrophages (moMϕ). Monocytes isolated from umbilical cord blood of term neonates and peripheral blood of healthy adults were differentiated in the absence or presence of hLF, and differentiation, apoptosis and phagocytosis were evaluated. Cytokine production, Toll-like receptor (TLR) signalling and activation marker expression were investigated upon activation with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge. We demonstrate that hLF-differentiated moMϕ exhibit decreased TLR-4 expression, TLR signalling, proinflammatory cytokine secretion and intracellular tumour necrosis factor (TNF)-α production. Investigation of differentiation markers, morphology and induction of apoptosis showed no alteration in lactoferrin-differentiated moMϕ. Taken together, hLF promote anergic/anti-inflammatory effects by TLR expression and pathway interference, resulting in a diminished proinflammatory moMϕ phenotype. The anergic/anti-inflammatory properties of hLF might contribute to the prevention of harmful TLR-mediated inflammatory disorders in the developing gut of premature infants.