The continuous increase in plastic production has resulted in increased generation of microplastic particles (MPs), and nanoplastic particles (NPs). Recent evidence suggests that nanoplastics may be a potent neurotoxin because they are able to freely cross the blood-brain barrier and enter the brain. Therefore, the cytotoxic effects of polystyrene nanoparticles (PS-NPs) on cellular systems of cerebral origin should be thoroughly investigated. The aim of the current study is to evaluate the cytotoxic potential of 25 nm PS-NPs on in vitro cultured cells such as primary astrocytes, neurons and their co-cultures established from the cerebral cortex of Wistar pups. The results show that PS-NPs are internalized in both neurons and astrocytes, inducing time- and concentration-dependent cytotoxic effects. However, quantification of fluorescence intensity indicates cell type-dependent differences in the efficiency of PS-NPs uptake. Astrocytes are several times more efficient at accumulating PS-NPs than neurons, and this is a phagocytosis-dependent process. Moreover, the high rate of PS-NPs internalization during prolonged exposure (72 h) promotes astroglial activation, as assessed by analysis of GFAP expression and immunocytochemical imaging. The results show that astroglia act as a cellular depot of PS-NPs to protect neurons. However, once the critical threshold is exceeded, astroglia become overactivated and can lose their protective functions. These results highlight the importance of further research on the mechanisms underlying nanoplastic-induced cellular toxicity, which may have implications for understanding the broader impact of plastic pollution on neurological functions.