The accumulation of nanoplastics (NPs) in crops has drawn global attention due to their potential exacerbation in human health through food chain transfer. The present study investigated the distribution, accumulation, and phytotoxicity of polystyrene (PS) NPs in radish and evaluated the potential risks of PS NPs to human health via a simulated INFOGEST model. PS NPs were mainly accumulated in the cortex and xylem of radish roots and primarily accumulated within the peels via direct adsorption onto tuber surfaces. Transcriptomic and metabolomic analyses revealed that exposure to PS NPs triggered plant defense systems by upregulating gene expression and metabolites involved in flavonoid biosynthesis as well as starch and sucrose metabolisms. However, the downregulation of genes associated with plant hormone signal transduction, as well as the biosynthesis of glucosinolates (the most valued compounds contributing to radish nutrition and flavor), and amino acids reduced crop yield and quality. Importantly, the investigations using a simulated INFOGEST model showed that PS NPs significantly reduced bioaccessibilities or index of nutritional quality (INQ) of amino acids and glucosinolates in the digesta of radish fruits, thereby compromising the nutritional value of radish. These findings further our understanding of the negative effects of NPs-contaminated crops on human digestive tract health.