Neonatal immune response is characterized by an uncompensated pro-inflammatory response that can lead to inflammation-related morbidity and increased susceptibility to infection. We investigated the effects of long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) pre-treatment on cytokine secretion to low-concentration endotoxin (lipopolysaccharide, LPS) in THP-1 monocytes and neonatal cord blood (CB) from healthy full-term infants. Pre-treatment of THP-1 cells, with either n-3 PUFA at 25 or 100 μM significantly reduced IL-6, IL-10, and IL-12 secretion while DHA, but not EPA, reduced TNF-α response to LPS. DHA inhibition was stronger compared to EPA and effective at the low concentration. The same concentrations of n-3 PUFAs inhibited IL-12 but not IL-10 cytokine response in whole CB from 9 infants pre-treated for 24 h. To assess clinical relevance for acute response to LPS, the effects of low-concentration DHA at 25 μM or 12.5 μM were assessed before and after LPS exposure of isolated CB mononuclear cells from 20 infants for 1 h. When added before or after LPS, physiologic DHA treatment produced significant concentration-dependent inhibition of TNF-α, IL-6, IL-1β, and IL-8 secretion. The results demonstrate prophylactic and therapeutic modulation of neonatal cytokine response to LPS and provide proof-of-concept that low-concentration administration of n-3 PUFA could attenuate or resolve neonatal inflammatory response.