Abstract: Xanthium spinosum is a noxious invasive weed that is spreading throughout Xinjiang Province. This plant threatens ecological safety, biodiversity, and agricultural development in affected areas. Because X. spinosum has shown strong adaptation to the divergent environment in the Ili River valley, samples of X. spinosum collected from this area were used as the research material in this study. To explore the allelopathic effects of this weed on vegetation succession and to determine the chemical mechanism of its noxious invasion, seeds of lettuce (Lactuca sativa var. longifolia) were treated with different concentrations of an aqueous extract of X. spinosum. The germination rate, germination energy, and germination index of lettuce seeds all decreased with increasing concentrations of the aqueous extract, and the germination speed was inhibited to different degrees. Compared with the control group, the seedlings treated with the extract at a concentration of 0.01 g/mL showed significantly inhibited root length. As the concentration of the aqueous extract increased, the root length of seedlings was significantly inhibited and there were significant differences in root length among the different treatments. The aqueous extract at a concentration of 0.08 g/mL showed the strongest inhibitory effect on seedling root length. The index of allelopathic effect (RI) decreased as the concentration of aqueous extract increased and the duration of the treatment extended. The results indicated that the aqueous extract of X. spinosum contained some allelopathic substances that inhibited seed germination and root elongation. The results of this study provide important information on the allelopathic effects of X. spinosum on vegetation succession and on the chemical mechanism of its invasion.