Profound alterations in the lipid profile of raft and non‐raft plasma membrane microdomains were found when RAW264.7 macrophages were supplemented with polyunsaturated fatty acids (PUFAs) in physiologically relevant concentrations. For the first time lipids in the detergent‐free isolated membrane domains of phagocytic immune cells were characterized by mass spectrometry. The extent of remodeling of the membrane lipids differed with different n3 and n6 PUFA supplements. The mildest effects were detected for α‐linolenic acid (LNA) and linoleic acid (LA), the C18 precursors of the n3 and n6 families, respectively. When the effects of highly unsaturated PUFAs were compared, eicosapentaenoic acid (EPA) caused more extensive restructuring of membrane lipids than docosahexaenoic acid (DHA) or arachidonic acid (AA). The supplements altered the lipid species composition of both the raft and non‐raft membrane fractions. The rafts containing elevated proportions of highly unsaturated lipid species may relocate sterically incompatible lipids and proteins originally belonging to this microdomain. Such effect was evident for sphingomyelin, which favored non‐rafts instead of rafts after EPA supplementation. The current work suggests that the different functional consequences found previously when supplementing macrophages with either EPA or DHA have their origin in the different effects of these PUFAs on membrane architecture. Profound alterations in the lipid profile of raft and non‐raft plasma membrane microdomains were found when RAW264.7 macrophages were supplemented with polyunsaturated fatty acids (PUFAs) in physiologically relevant concentrations. The supplements altered the lipid species composition of both the raft and non‐raft membrane fractions. Furthermore, the current work suggests that the different functional consequences found previously when supplementing macrophages with either EPA or DHA have their origin in the different effects of these PUFAs on membrane architecture.