Root border cells (RBCs) are a group of cells that exhibit distinct gene expression patterns. They are detached from the tips of plant roots and distributed into the soil environment. Similar to mammalian immunocytes, RBCs create barriers within plant roots, thereby playing a significant role in plant defense mechanisms. Chenopodium ambrosioides, an invasive plant, exerts a substantial impact on neighboring plants, altering their morphology, physiology, and gene expression. The aqueous extract of C. ambrosioides inhibits the germination of seeds and growth of seedlings in Fagopyrum tataricum, whereas the root border cells alleviate the adverse effects of allelochemical stress. To investigate the molecular mechanism underlying this mitigating effect, we examined the changes in the levels of reactive oxygen species (ROS), superoxide anion (O₂^-), and malondialdehyde (MDA), as well as the activity of antioxidant enzymes (Superoxide dismutase, SOD; Peroxidase, POD; Catalase, CAT), in the group with preserved root border cells and the group with removed root border cells, following treatment with aqueous extracts of C. ambrosioides. The results demonstrated that treatment with the aqueous extract of C. ambrosioides significantly increased the levels of the ROS, O², MDA, and the activities of antioxidant enzymes (POD and CAT) in root tips of Fagopyrum tataricum. The RBC-removing group exhibited a higher increase compared to the RBC-preserving group. Pathway analysis revealed the notable alterations in phenylpropanoid synthesis, alpha-linolenic acid metabolism, flavonoid synthesis, and glutathione metabolism in both the RBC-preserving and RBC-removing groups following treatment with the aqueous extract of C. ambrosioides. Specifically, the phenylpropanoid synthesis pathway was primarily suppressed, with a less degree of inhibition observed after removing RBCs. The expression levels of steryl ester hydrolase (TGL4) and phospholipase A1 (DAD1) in the alpha-linolenic acid metabolism pathway were significantly upregulated, while no changes were observed in the RBC-removing group. TGL4 and DAD1 were responsible for catalyzing the conversion of phospholipid choline into alpha-linolenic acid, which provided abundant substrates for inhibiting plant growth and enhancing resistance to jasmonic acid (JA). Moreover, the synthesis pathways associated with cutin, suberin, and wax, which contributed to root hydrophobicity, were significantly altered exclusively in the RBC-removing group. These findings suggest that root border cells strive to achieve a balance between growth and defense by influencing the synthesis of stress-resistant substances (phenylpropanoid pathway), antioxidant enzymes, and non-enzymatic antioxidant systems (flavonoids and glutathione), while also enhancing the expression of JA. This, in turn, helps mitigate the allelopathic stress caused by water-soluble substances of C. ambrosioides. Finally, the composition of the aqueous extracts was complex and the component plays a major role in the allelopathy is still to be confirmed by further research.