The internalization of A.fumigatus into lung epithelial cells is a critical process for the invasion and immune evasion of A.fumigatus in the host.Gliotoxin is the major and the most potent toxin produced by A.fumigatus, however, its role in the interaction between A.fumigatus and lung epithelial ceils is largely unknown.Here we reported that gliotoxin enhanced the internalization of conidia of A.fumigatus into A549 lung epithelial cells but inhibited the phagocytosis of J774 macrophage in a dose-dependent manner.Deletion of gliotoxin-producing regulating gene glipsuppressed the internalization of A.fumigatus conidia into epithelial cells,which can be rescued by the addition of gliotoxin.Treatment of gliotoxin had not effect on cell viability under 100 ng/ml but suppressed ROS production in a dose-dependent manner.Gliotoxin stimulated the phospholipase D activity of A549 cells largely at the concentration of 50 ng/ml and this stimulation was blocked by pretreatment with PLDl-specific inhibitor but not PLD2-specific inhibitor.Gliotoxin induced the cell morphological change of A549 cells combining with great actin cytoskeleton rearrangement and moderate alteration of the distribution of phospholipase D.These data indicated that gliotoxin might play an important role in A.fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement.