Nanoplastics, arising from the fragmentation of plastics into environmental pollutants and specialized commercial applications, such as cosmetics, have elicited concerns due to their potential toxicity. Evidence suggests that the oral ingestion of nanoplastics smaller than 100 nm may penetrate the brain and induce neurotoxicity. However, comprehensive research in this area has been hampered by technical challenges associated with the detection and synthesis of nanoplastics. This study aimed to bridge this research gap by successfully synthesizing fluorescent polystyrene nanoplastics (PSNPs, 30-50 nm) through the incorporation of IR-813 and validating them using various analytical techniques. We administered PSNPs orally (10 and 20 mg/kg/day) to mice and observed that they reached brain tissues and induced cognitive dysfunction, as measured by spatial and fear memory tests, while locomotor and social behaviors remained unaffected. In vitro studies (200 μg/mL) demonstrated a predominant uptake of PSNPs by microglia over astrocytes or neurons, leading to microglial activation, as evidenced by immunostaining of cellular markers and morphological analysis. Transcriptomic analysis indicated that PSNPs altered gene expression in microglia, highlighting neuroinflammatory responses that may contribute to cognitive deficits. To further explore the neurotoxic effects of PSNPs mediated by microglial activation, we measured endogenous neuronal activity using a multi-electrode array in cultured hippocampal neurons. The application of conditioned media from microglia exposed to PSNPs suppressed neuronal activity, which was reversed by inhibitors of microglial activation. Our findings offer detailed insights into the mechanisms by which nanoplastics damage the brain, particularly emphasizing the potential environmental risk factors that contribute to cognitive impairment in neurodegenerative diseases.