Tight junctions of epithelial cells determine epithelial membrane integrity and play an important role in selective paracellular permeability to ions and macromolecules. In this work, we investigated the effect of one of n-3 series polyunsaturated fatty acids, eicosapentaenoic acid (EPA) on the phospholipid composition of membrane microdomains of tight junctions. After treated by EPA, membrane microdomains of tight junctions were isolated by discontinuous sucrose density gradient ultracentrifugation, and raft phospholipids were extracted. The PE, PI, PS, PC and SM were separated and determined by high-performance liquid chromatography/quadrupole-linear ion trap mass spectrometry (HPLC Qtrap-MS), and were further identified by HPLC–MS/MS. It was found that EPA altered the fatty acyl substitution of phospholipids that constituted membrane microdomains of tight junctions by enriching the unsaturated fatty acyl chains of the phospholipids. It provides a new visual angle to explaining the intracellular mechanism involved in n-3 polyunsaturated fatty acids (PUFAs) modulation of intestinal tight junction barrier.