Nanoplastics (NPs) have been demonstrated the ability to penetrate plant roots and cause stress. However, the extent of NPs penetration into various root tissues and the corresponding plant defense mechanisms remain unclear. This study examined the penetration and accumulation patterns of polystyrene nanoplastics (PS-NPs) in different cell types within rice roots, and explored how the roots quickly modify their cell wall structure in response. The findings showed that fully developed sclerenchyma cells in rice roots effectively prevented the invasion of PS-NPs. Meanwhile, PS-NPs triggered the accumulation of lignin and suberin in specific cells such as the exodermis, sclerenchyma, and xylem vessels. PS-NPs at a concentration of 50mg/L increased cell wall thickness by 18.6%, 21.1%, and 22.4% in epidermis, exodermis, and sclerenchyma cells, respectively, and decreased root hydraulic conductivity by 14.8%. qPCR analysis revealed that PS-NPs influenced the cell wall synthesis pathway, promoting the deposition of lignin and suberin monomers on the secondary wall through the up-regulation of genes such as OsLAC and OsABCG. These results demonstrate that PS-NPs can induce cell type-specific strengthening of secondary walls and barrier formation in rice roots, suggesting the potential role of plant secondary wall development in mitigating NPs contamination risks in crops. Environmental ImplicationRice is a crucial food crop, necessitating research on the infiltration of nanoplastics (NPs) pollutants into rice plants and the roots’ resistance mechanisms. For the first time, we proved that NPs hasten the accumulation of lignin and suberin in rice roots. The fortified outer barrier of roots, constituted by the exodermis and sclerenchyma, effectively blocked NPs invasion but also decreased root hydraulic conductivity. Nevertheless, the fragile root tips could be a vulnerable site for NPs invasion, warranting attention. These findings provide valuable insights for further exploration of crop resistance mechanisms to NPs in field ecosystems and for promoting agricultural sustainability.