Perennial ryegrass (Lolium perenne L.) was planted in uranium-contaminated soil mixtures supplemented with different amounts of citric acid to investigate the defense strategies of perennial ryegrass against U and the enhanced mechanism of citric acid on the remediation efficiency in the laboratory. The uranium content in the plant tissues showed that the roots were the predominant tissue for uranium accumulation. In both root and shoot cells, the majority of U was located in the cell wall fraction. Furthermore, antioxidant enzymes were also stimulated when exposed to U stress. These results suggested that perennial ryegrass had evolved defense strategies, such as U sequestration in root tissue, compartmentalization in the cell wall, and antioxidant enzyme systems, to minimize uranium stress. For an enhanced mechanism, the optimal concentration of citric acid was 5mmol/kg, and the removal efficiency of U in the shoots and roots increased by 47.37% and 30.10%, respectively. The treatment with 5mmol/kg citric acid had the highest contents of photosynthetic pigment and soluble protein, the highest activity of antioxidant enzymes, and the lowest content of MDA (malondialdehyde) and relative electrical conductivity. Moreover, the TEM (transmission electron microscope) results revealed that after 5mmol/kg citric acid was added, the cell structure of plant branches partially returned to normal, the number of mitochondria increased, chloroplast surfaces seemed normal, and the cell wall became visible. The damage to the cell ultrastructure of perennial ryegrass was significantly alleviated by treatment with 5mmol/kg citric acid. All the results above indicated that perennial ryegrass could accumulate uranium with elevated uranium tolerance and enrichment ability with 5mmol/kg citric acid.