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Nanoplastics pollution is a rising environmental concern whose impacts on biodiversity and human health are far from being understood. This is particularly salient in aquatic ecosystems, where the majority of species depend on external fertilization for reproduction. Here we evaluated the effects of a short-term exposure to engineered polystyrene nanoplastics (NPs) in the zebrafish germline to further explore their impact on reproduction. To this end, zebrafish (Danio rerio) were exposed to 5 mg/L of 45 nm polystyrene (PS)-NPs via water for 96 h. We show that, in males, nanoplastics induced testicular histological alterations with abnormal sperm clustering and chromatin compaction, resulting in viable spermatozoa but with reduced motility. Moreover, in females we observed an alteration in oocyte stages frequencies during oogenesis, possibly reflecting alterations in oocyte growth. RNA-sequencing analysis in male testis links nanoplastic induced alterations in the expression of genes involved in chromatin structure, meiosis and DNA double-strand break formation and repair progression, and gametes recognition. Flow cytometry analysis revealed that the observed effects in males were directly due to nanoplastics penetrating the testicular barrier and being internalized within germline cells. Overall, our results demonstrate that acute exposure to NPs can compromise reproductive fitness, underscoring the environmental and health impacts of NPs pollution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)