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Copper oxide nanoparticles (CuO NPs) are widely used as a fungicide in agriculture. The application of CuO NPs in agriculture affects the growth of rice and metal accumulation in rice. However, the mechanism of CuO NPs on arsenic (As) accumulation in rice remains unclear. In this study, a hydroponic culture was produced to investigate the mechanism of the effect of 50 and 100 mg L−1 CuO NPs on As accumulation in rice. Our results showed that CuO NPs decreased As(III/V) accumulation in the roots and shoots by adsorbing As(III/V), oxidizing of As(III) on the surface, and thickening the root cell wall. The addition of CuO NPs regulated the expression of the OsNIP1;1, OsHAC1;1, and OsHAC4 genes, which decreased As(III) transport and promoted As(V) reduction in the roots. Moreover, when CuO NPs were co-exposed to As, a negative correlation between the concentration of Cu and As in rice was also found in our study. However, CuO NPs significantly increased Cu accumulation in rice and constrained the rice growth. In conclusion, CuO NPs might be a promising way to decrease As accumulation in rice, but the negative effects such as growth inhibition should be further considered. Therefore, the application of CuO NPs in rice plants should take a more restrained approach. [ABSTRACT FROM AUTHOR]