In this research, the lettuce high-temperature-sensitive variety Beisan San 3 was used as a test material. The effects of exogenous spermidine (Spd) on membrane lipid peroxidation, the antioxidant system, the ascorbic acid-glutathione (AsA-GSH) system and the glyoxalase (Glo) system in lettuce seedlings under high-temperature stress were studied by spraying either 1 mM spermidine or ionized water as a control. The results showed that, under high-temperature stress, the growth of lettuce seedlings was weak, and the dry weight (DW) and fresh weight (FW) were reduced by 68.9% and 82%, respectively, compared with those of the normal-temperature controls. In addition, the degree of membrane lipid peroxidation increased, and the reactive oxygen species (ROS) level increased, both of which led to a significant increase in malondialdehyde (MDA) content and lipoxygenase (LOX) activity. Under high-temperature stress, the activity of superoxide dismutase (SOD) decreased, the activities of peroxidase (POD) and catalase (CAT) increased first but then decreased, and the activity of ascorbic acid peroxidase (APX) decreased first but then increased. Glutathione reductase (GR) activity, ascorbic acid (AsA) and glutathione (GSH) content showed an upward trend under high-temperature stress. The activities of glyoxalase (GloI and GloII) in the lettuce seedling leaves increased significantly under high-temperature stress. In contrast, the application of exogenous Spd alleviated the oxidative damage to the lettuce seedlings, which showed a decrease in MDA content and LOX activity and an increase in SOD, POD, CAT, APX, GR, GloI, and GloII activities. In addition, the antioxidant AsA and GSH contents also increased to varying degrees. It can be seen from the results that high temperature stress leads to an increase in the level of ROS and cause peroxidation in lettuce seedlings, and exogenous Spd can enhance the ability of lettuce seedlings to withstand high temperature by enhancing the antioxidant system, glyoxalase system and AsA-GSH cycle system.