Polyunsaturated fatty acids, including docosahexaenoic acid (DHA, 22:6n-3), modulate immune responses and exert beneficial immunosuppressive effects, but the molecular mechanisms inhibiting T-cell activation are not yet elucidated. Lipid rafts have been shown to play an important role in the compartmentalization and modulation of cell signaling. We investigated the role of DHA in modulating the lipid composition in lipid rafts and membrane subdomain distribution of interleukin-2 (IL-2) receptor signaling molecules. We found that DHA altered lipid components of rafts and modified the IL-2-induced Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway by partially displacing IL-2 receptors from lipid rafts. We fractionated plasma membrane subcellular compartments and discovered that certain amounts of STAT5a and STAT5b existed in detergent-resistant plasma membrane fractions of T-cells. After DHA treatment, STAT5a and STAT5b were not detected in lipid raft fractions and were located in detergent-soluble fractions. These data demonstrate for the first time that DHA alters the lipid composition of membrane microdomains and suppresses IL-2 receptor signaling in T-cells. Thus, our data provide evidence for a functional modification in lipid rafts by DHA treatment and explain PUFA-mediated immunosuppressive effects.