The invasive plant Chenopodium ambrosioides L. exerts strong allelopathic effects on native crops, while root border cells (RBCs) of recipient plants can rapidly respond to such allelopathic stress by forming a protective layer on the outer surface of the root tip within a short period, thereby maintaining normal root tip metabolism. To investigate the molecular mechanisms underlying this phenomenon, maize (Zea mays L.) was used as the experimental model. The study employed length measurement and high-performance liquid chromatography (HPLC) to determine changes in root tip length and auxin (IAA) content in maize under the stress of C. ambrosioides volatiles and its major component, α-terpinene, in both RBCs-retained and RBCs-removed groups. Additionally, paraffin sectioning combined with microscopy was used to observe internal structural changes in the root tips. Finally, transcriptome sequencing was performed to analyze the overall impact of this process on auxin metabolism. The results demonstrated that under C. ambrosioides volatile stress, the number and activity of RBCs decreased significantly compared to the control group, with the inhibitory effects becoming more pronounced as treatment duration and concentration increased. The comprehensive allelopathic effect index of C. ambrosioides volatiles and α-terpinene on maize RBCs was ranked as C. ambrosioides volatiles (0.191) > α-terpinene (0.172). Upon RBCs removal, the root cap of the recipient plant was severely damaged, IAA content was reduced, mature zone cells within the root tip became smaller, and root tip length decreased. Under α-terpinene treatment, the endodermis and pericycle cell layers were notably thickened. Transcriptomic analysis revealed that allelochemical stress affected the processes of auxin synthesis, transport, and signaling transduction. Volatile treatment upregulated IAA biosynthesis genes, such as YUCCA and TAR2. However, after RBCs removal, genes associated with polar auxin transport, such as the PIN family, were upregulated, along with negative regulators of IAA signaling transduction, including AUX/IAAs and GH3s. This led to a reduction in IAA accumulation at the root tip and suppression of IAA function. These findings indicate that under C. ambrosioides volatile stress, RBCs respond to the stress by reducing their own activity and cell numbers, thereby participating in the regulation of IAA polar transport and signaling transduction gene expression. By maintaining normal IAA polar transport and signaling transduction, RBCs achieve a balance between regulating maize root tip development and responding to allelopathic stress.