Zinc oxide nanoparticles (ZnO NPs) are widely used to manufacture textile fibers, synthetic rubber, and paint. However, crop yields and quality are threatened by the increased use of metallic NPs in industry, which has resulted in their accumulation in agricultural land. Many studies have shown that plants defend against biotic and abiotic stresses through the activities of metabolites and hormones. However, whether glucosinolates (GSs) are involved in plant responses to ZnO NP-related stress remains unknown. In this study, wild-type (WT) and GS mutant (myb28/29 and cyp79B2/B3) Arabidopsis plants were subjected to ZnO NP stress to address this question. Our results showed that exposure to ZnO NPs promoted GS accumulation and induced the relative messenger RNA (mRNA) expression levels of GS biosynthesis-related genes. Moreover, ZnO NP treatment adversely affected root length, the number of lateral roots, chlorophyll contents, and plant biomass. Importantly, our results showed that root growth, chlorophyll contents, and plant biomass were all decreased in the GS mutants compared with those in WT plants. Overall, our results showed that WT plants tolerated ZnO NP-induced stress more efficiently than the GS mutants, suggesting that GSs are involved in plant resistance to ZnO NP-induced toxicity.