© 2014 Elsevier Ltd. Introduction Dietary supplementation with omega-3 long chain polyunsaturated fatty acids (n-3 PUFAs) may exert benefits in pregnancy through inhibition of placental inflammation. However, studies on the anti-inflammatory effects of n-3 PUFAs in the placenta are lacking. We compared the cytokine responses of human placental explants in vitro after 4 days pre-incubation with either: a) individual n-3 or n-6 PUFAs (20 μM), or b) physiologically relevant combinations of low, medium or high n-3 or n-6 PUFA concentrations. Methods Placental cytokine (IL-6 and TNF-α) mRNA expression and protein production were assessed at 4 h and 12 h, respectively. Cytokine and fatty acid concentrations were also measured in placentas delivered at term by women who ingested either low (n = 12) or high (n = 10) amounts of fish/fish oil in the month prior to delivery. Results Pre-exposure to n-3 PUFAs as individual fatty acids results in reduced placental IL-6 production (P <0.05), whereas exposure to complex fatty acid mixtures enriched in n-3 PUFAs (high n-3:n-6 ratios) results in a significant stimulation of IL-6 production (P <0.05). There were no differences in placental n-3 or n-6 PUFA levels between women with either high or low dietary fish oil intake and no differences in cytokine expression. Discussion In summary, data from our complex lipid explant model and an observational cohort study do not support a role for n3 PUFAs in the suppression of pro-inflammatory cytokine expression in the human placenta. Results from studies of placental tissues exposed to single n-3 and n-6 PUFAs should be interpreted with considerable caution.