Nanoplastics (NPs) are derived from microplastics and may cause health problems. We previously showed that 100 nm polystyrene (PS)-NPs enter cells, including mouse embryonic fibroblasts (MEFs), and their intracellular accumulation induces inflammatory and oxidative stress. Moreover, PS-NP uptake was found to occur via endocytosis, and they accumulated mostly at the juxtanuclear position, but never within the nucleus. We speculated that PS-NPs were cleared from cells when they were no longer exposed to PS-NPs. However, the effects of PS-NPs on the cellular machinery remain unknown. The accumulation of PS-NPs at the juxtanuclear position may be due to retrograde transport along microtubules. To confirm this, we treated PS-NP-exposed MEFs with inhibitors of histone deacetylase 6 (HDAC6), dynein, or microtubule polymerization and found greatly diminished intracellular and juxtanuclear accumulation. Moreover, rapid clearance of PS-NPs was observed when MEFs were treated with an HDAC6 inhibitor. PS-NPs were removed by exocytosis, as confirmed by treatment with an exocytosis inhibitor. Furthermore, inhibiting the retrograde transport of PS-NPs alleviated the activation of the antioxidant response pathway, inflammatory and oxidative stress, and reactive oxygen species generation. In summary, inhibition of the retrograde transport of non-biodegradable PS-NPs leads to their rapid export by exocytosis, which may reduce their cytotoxicity.