Abstract Objective The aim of the present study was to examine the immune-modulating effect of two different fat blends enriched with a low dose of anti- or proinflammatory polyunsaturated fatty acids on the fatty acid status and subsequently on the immune response of healthy volunteers. Methods Thirty healthy volunteers were randomly assigned to group A (anti-inflammatory blend rich in polyunsaturated fatty acids: α-linolenic acid, 240 mg/d; eicosapentaenoic acid, 120 mg/d; stearidonic acid, 49 mg/d; and γ-linolenic acid, 73 mg/d) or group B (arachidonic acid, 40 mg/d; containing an inflammatory fat blend) for a 2-wk dietary supplementation period. Concentrations of interleukin-8, interleukin-10, tumor necrosis factor-α, prostaglandins E 1 and E 2, and leukotriene B 4 were investigated before, after 2 wk of supplementation, and 2 wk after stopping supplementation using a whole blood ex vivo lipopolysaccharide-stimulation assay. Results Plasma concentrations of α-linolenic acid and eicosapentaenoic acid were significantly increased in group A. In addition, dietary fat blends influenced eicosapentaenoic acid concentration in erythrocyte membranes. Supplementation of the fat blends resulted in contrasting effects on the expression of lipid mediators and cytokines after ex vivo lipopolysaccharide stimulation. Release of prostaglandin E 1 and leukotriene B 4 were significantly decreased in group A, whereas prostaglandin E 2 and interleukin-10 concentrations were significantly increased in group B. No effect on interleukin-8 or tumor necrosis factor-α release was found after supplementation with either fat blend. Conclusions These results show an immune-modulating effect of a low-dose dietary polyunsaturated fatty acid supplementation. However, further studies regarding fat-blend composition and period of supplementation in patients with inflammatory conditions are required.