Epigenetic regulation plays an important role in regulating the transgenerational toxicity of pollutants. However, underlying mechanism of microRNAs (miRNAs) in regulating transgenerational nanoplastic toxicity remains largely unclear. We aimed to determine miRNA-mediated mechanism for induction of transgenerational nanoplastic toxicity. In Caenorhabditis elegans, although germline RNAi of both mir-240 and mir-36 suppressed polystyrene nanoparticle (PS-NP) toxicity, exposure to PS-NP (1-100 μg/L) only increased mir-240 expression. Transgenerational increase in mir-240 expression was observed after PS-NP exposure at P0 generation (P0-G), and germline RNAi of mir-240 suppressed transgenerational PS-NP toxicity. Among predicted target genes of mir-240 in the germline, exposure to PS-NP (1-100 μg/L) decreased rab-5 and rab-6.2 expressions, whereas germline RNAi of mir-240 only increased rab-5 expression in PS-NP exposed nematodes. Transgenerational decrease in rab-5 expression was detected after PS-NP exposure at P0-G, and germline RNAi of rab-5 strengthened transgenerational PS-NP toxicity. Moreover, the resistance of mir-240(RNAi) to transgenerational PS-NP toxicity in inhibiting locomotion behavior and in reducing brood size was inhibited by germline RNAi of rab-5. Among secreted ligands, germline RNAi of rab-5 increased expressions of genes encoding insulin peptides (ins-3, ins-39, and daf-28), FGF ligand (egl-17), and Ephrin ligand (efn-3) in PS-NP exposed nematodes and their corresponding receptor genes (daf-2, egl-15, and vab-1) in offspring of PS-NP exposed nematodes. Therefore, increase in germline mir-240 mediated transgenerational PS-NP toxicity through insulin, FGF, and Ephrin signals by affecting its target of RAB-5. Our data provided important involvement of germline microRNA in mediating nanoplastic toxicity across multiple generations in organisms.