AbstractBackgroundMicroplastic pollution increasingly affects human health. Polystyrene nanoparticles (PS‐NPs) and microplastics (PS‐MPs) may impair Sertoli cells (SCs), vital for male fertility.ObjectivesTo compare PS‐NPs (80 nm)/PS‐MPs (8 µm) toxicity on rat SCs, focusing on oxidative stress, apoptosis, epithelial barrier integrity, endocytosis pathways, and miRNA‐mediated ceRNA networks.Materials/methodsSCs viability was assessed via CCK‐8. Trans‐epithelial electrical resistance (TER) was measured to assess the epithelial barrier function. Particle internalization was evaluated by confocal microscopy and flow cytometry. Endocytosis was detected by using specific inhibitors. Whole‐transcriptome sequencing profiling identified differential expression of miRNAs, lncRNAs, circRNAs, and mRNAs, with subsequent ceRNA network construction. Pro‐inflammatory factors and apoptosis were detected by RT‐qPCR and flow cytometry, respectively.ResultsAt 100 µg/mL, PS‐NPs reduced cell viability to 77% versus 94% for PS‐MPs (p < 0.05) and decreased TER by 81% versus 63% for PS‐MPs (p < 0.01). PS‐NPs were internalized via clathrin‐ and caveolin‐dependent pathways, while PS‐MPs remained extracellular. RNA‐seq revealed PS‐MPs activated inflammation pathways (Ank3/Daxx), while PS‐NPs triggered oxidative stress and apoptosis pathways (Map2k4/Grin2a). PS‐NPs induced higher apoptosis (17% vs. 9.3%, p < 0.01), Reactive oxygen species (3.7‐fold vs. 1.87‐fold), and more severe catalase activity reduction (67% vs. 17%, p < 0.01) compared to PS‐MPs.Discussion and conclusionPS‐NPs pose greater toxicity to SCs than PS‐MPs due to cellular internalization, disrupting barrier integrity via oxidative stress/apoptosis. PS‐MPs primarily trigger extracellular inflammation. Distinct ceRNA networks underpin their differential mechanisms. These results highlight risks of environmental microplastic fragmentation into nanoplastics, emphasizing the need for further research on microplastic impacts on male fertility.