Abstract <p>Reasonable research on the structural modification of natural products is of great value for the development of anti-tumor drugs. In this study, using the natural active compound ergosterol peroxide as the leading structure, eight ergosterol peroxide-3-maleate amide derivatives were designed and synthesized. Their cytotoxicity <i>in vitro</i> was evaluated against human lung cancer A549 cell line, breast cancer MCF-7 cell line, hepatocellular carcinoma HepG2 cell line, and human embryonic lung fibroblast MRC-5 cell line. Among them, (3β,5α,8α,22<i>E</i>)-5,8-epidioxyergosta-6,22-dien-3-yl-4-(dimethylamino)-4-oxobut-2-enoate exhibited more potent <i>in vitro</i> antiproliferative activity against A549 cells, with a half-maximal inhibitory concentration (IC₅₀) of 4.26 μM. Further study of the mechanisms suggests that it promoted reactive oxygen species (ROS) production in a dose-dependent manner and had a total apoptosis rate of 24.82% at a concentration of 8 μM in A549 cells. These findings suggest that the generation of ROS and the apoptosis may be two key indicators of oxidative stress induced by (3β,5α,8α,22<i>E</i>)-5,8-epidioxyergosta-6,22-dien-3-yl-4-(dimethylamino)-4-oxobut-2-enoate. We further explored the molecular docking between thioredoxin reductase (TrxR) protein and (3β,5α,8α,22<i>E</i>)-5,8-epidioxyergosta-6,22-dien-3-yl-4-(dimethylamino)-4-oxobut-2-enoate, discovering a strong binding affinity between them. Based on these observations, it is suggested that this new compound may regulate cellular redox homeostasis in A549 cells by inhibiting TrxR protein expression and increasing ROS production, thereby enhancing its pro apoptotic effect.</p>