Abstract: To explore how low \mathrmNO_3^- stress affects the apoplastic pathway transport in tobacco root, two low \mathrmNO_3^- stress treatments were implemented at concentrations of 0.01 and 0.1 mmol/L,while 4.0 mmol/L \mathrmNO_3^- treatment served as the control. The flue-cured tobacco variety Zhongyan100 was employed as the experimental material. Various parameters including tobacco seedling development, root morphology, transpiration rate, apoplastic pathway transport, endodermal suberization, and gene expression related to apoplastic barrier formation were analyzed. The root/shoot of low \mathrmNO_3^- stress (0.01 mmol/L and 0.1 mmol/L) was significantly higher than that of the control, with increases of 267% and 125%, respectively; The transpiration capacity of low \mathrmNO_3^- stress was significantly lower than that of the control, with decreases of 176% and 114%, respectively; The PTS fluorescence intensity of low \mathrmNO_3^- stress is significantly lower than that of the control, with the decrease of 31% and 27%, respectively. The PTS concentrations in xylem sap and shoot of low \mathrmNO_3^- stress exhibited lower than that of the control, with the decrease of 105%, 91% and 103%, 75%, respectively. Moreover, low \mathrmNO_3^- stress induced early endodermal suberization of tobacco roots, with suberin deposition observed nearer to the root tip. This stress condition meanwhile upregulated gene expression associated with apoplastic barrier formation. In summary, low \mathrmNO_3^- stress-induced endodermal suberization inhibited the apoplastic pathway transport in tobacco roots.