【Objective】Micro- and nanoplastics have emerged as pervasive contaminants in terrestrial ecosystems. However, current research remains disproportionately focused on aquatic environments and food crops, leaving a significant knowledge gap regarding their effects on economically important non-food cash crops like tobacco, which possess high economic value and complex secondary metabolic pathways. This study systematically investigates the physiological and metabolic responses of Nicotiana benthamiana to root exposure of polystyrene nanoplastics (PS-NPs), with a particular focus on organ-specific adaptations in carbon and nitrogen metabolism under stress. Understanding these mechanisms is critical for ecological risk assessment and for safeguarding the productivity and quality in non-food cash crop systems, which have been largely neglected in the current nanoplastic research paradigm. 【Method】We employed a dual experimental approach integrating both pot cultivation and hydroponic systems to comprehensively evaluate PS-NPs effects on N. benthamiana seedlings. This integrated design enabled us to distinguish direct particle-plant interactions under controlled hydroponic conditions from more complex soil-mediated effects in pot environments. We employed metabolomics analysis coupled with detailed physiological analyses, including oxidative stress markers, antioxidant enzyme activities, and biomass measurements, to unravel the metabolic and defense networks activated under PS-NPs stress. 【Result】Pot experiments revealed a clear dose-dependent inhibition of plant growth, with PS-NPs concentrations of 150, 500, and 800 mg·kg-1 reducing plant height by 18.80%, 29.42%, and 30.67%, respectively. Hydroponic exposure induced even more striking morphological alterations, characterized by significant shoot suppression accompanied by a remarkable 43.52% and 47.20% increase in root elongation at 50 and 150 μg·mL-1. Paradoxically, the shoot fresh weight increased while dry weight accumulation was markedly reduced, indicating fundamental disruptions in carbon partitioning and structural biomass synthesis. Physiological analyses demonstrated severe oxidative stress in N. benthamiana roots, evidenced by elevated hydrogen peroxide and malondialdehyde levels alongside significantly enhanced superoxide dismutase activity, indicating activation of the antioxidant defense system. Metabolomic profiling identified extensive perturbations across multiple pathways, particularly in amino acid metabolism, carbohydrate dynamics, and organic acid transformation. It indicated that PS-NPs exposure disrupted central carbon metabolism, including carbon metabolism, galactose metabolism, and energy production pathways through glycolysis and oxidative phosphorylation. Moreover, N. benthamiana roots exhibited substantial downregulation of critical TCA cycle intermediates, including citrate and α-ketoglutarate, coupled with reduced glycolytic intermediates such as glucose-6-phosphate and fructose-6-phosphate, while simultaneously accumulating compatible solutes like isoleucine and valine. This result indicates strategic reallocation of nitrogen resources toward osmotic protection and fundamental defense mechanisms. Conversely, N. benthamiana leaves implemented an efficient carbon sequestration strategy, accumulating hexose phosphates and soluble sugars, and upregulating the biosynthesis of specialized defensive compounds, including flavonoid secondary metabolites and non-protein amino acids, demonstrating organ-specific metabolic specialization. Importantly, nitrogen metabolism of N. benthamiana leaves also shifted toward active defense and signal transduction. The pronounced upregulation of 4-aminobutyric acid (GABA) and its derivative 2,4-diaminobutyric acid marked the activation of the GABA pathway, a pivotal stress-response pathway. This pathway plays a crucial role in the reconstruction of carbon and nitrogen balance, and also assumes core functions in mitigating oxidative stress and regulating signal transduction within the N. benthamiana defense network. 【Conclusion】 This study demonstrates that PS-NPs root exposure initiates a complex adaptive response in N. benthamiana seedlings, characterized by inhibited shoot growth and dry matter accumulation, and stimulated root elongation as a stress-avoidance mechanism. PS-NPs root exposure also induced oxidative damage and triggered the comprehensive reorganization of metabolic networks. The research reveals an organ-specific defense strategy wherein roots prioritize immediate survival through osmotic adjustment and basic defense, while the leaves activate advanced chemical defense pathways, coordinated in part through GABA-mediated signaling. This study provides novel mechanistic insights into the metabolic adaptation of plants under nanoplastic stress and offers an important scientific basis for assessing the potential ecological risks of micro- and nanoplastics in terrestrial environments.