Nanoplastic exposure could cause toxicity to Caenorhabditis elegans at various aspects. Nevertheless, the effects of chronic exposure to nanoplastics remain largely unclear in nematodes. In this study, we employed C. elegans as an animal model to determine the effects of nanopolystyrene (30 nm) exposure from adult day-1 for 8-day. After the exposure, only 1000 μg/L nanopolystyrene reduced the lifespan. In contrast, nanopolystyrene ≥1 μg/L decreased locomotion behavior and activated oxidative stress. Meanwhile, in 10 μg/L nanopolystyrene exposed nematodes, both expression of SOD-3, a Mn-SOD, and autophagy induction as indicated by LGG-1:GFP expression were significantly increased. RNAi knockdown of daf-2 encoding an insulin receptor enhanced the autophagy induction, and RNAi knockdown of daf-16 encoding a FOXO transcriptional factor in insulin signaling pathway suppressed the autophagy induction in 10 μg/L nanopolystyrene exposed nematodes. Moreover, DAF-16 acted upstream of LGG-1, an ortholog of Atg8/LC3, to regulate the toxicity of nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior at adult day-9. Our data implied the potential toxicity of chronic exposure to nanoplastics at predicted environmental concentrations on organisms.