Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have immunosuppressive properties and beneficial effects towards autoimmune diseases. ω-3 PUFA supplementation effects on activation responses from overweight/obese women lymphocytes in a double blind, randomized, and controlled trial were evaluated. Forty overweight/obese women, about 57 years old, were recruited and randomized into two groups: supplemented with soy lecithin or fish oil (2g/day) for three months. Anthropometry, plasma lipid profile, fasting blood glucose levels, lymphocyte phenotypes (Th1/Th2/Th17) and proliferative capacity were performed. w-3 PUFA supplementation decreased fasting blood glucose, total cholesterol levels, percentages of Th1/Th2/Th17 cells and lymphocyte proliferation. Soy lecithin promoted a slight decrease in Th1 and proliferative responses in cells stimulated with concanavalin A. To investigate if ω-3 PUFAs modulate lymphocyte activation through changes in cell metabolism, extracellular glucose metabolism was measured in lymphocytes isolated from healthy volunteers blood and incubated with EPA and DHA in vitro, that in the presence of PMA/ionomycin exhibited a switch from mitochondrial towards a high glycolytic metabolism phenotype. The treatment partially prevented this switch as indicated by reduction in glucose uptake, glycolytic capacity and reserve. ω-3 PUFAs effects on lymphocyte energy metabolism and signalling contribute to the immunosuppressive actions of these fatty acids in humans.