CuO engineered nanoparticles (NPs) are of increasing concern due to their extensive use in daily life and adverse effect on aquatic organisms. The investigations on the toxicity of CuO NPs to aquatic plants through uptake from roots versus fronds are limited. This paper discusses the interactions of CuO NPs with Lemna minor, a floating plant. After CuO NPs (150 μg L-1) exposure for 7 days, the frond number, frond surface area and dry weights of whole plants significantly decreased by 32%, 47% and 33%; the responses were dose-dependent. Microscopy imaging showed that the epidermis was severely damaged in fronds, edges were severely sloughed off and cell integrity was damaged in roots. Shrinkage of both chloroplast and starch grains were observed in the frond cells. Internalization of CuO NPs in root and frond cells during CuO NPs (1 mg L-1) exposure was confirmed with the root Cu levels of Lemna minor being three times higher than the fronds by using transmission electron microscopy and flame atomic absorption spectrophotometry. Reactive oxygen species, mainly H2O2 (increased by 56%) and ·OH (increased by 57%), accumulated in Lemna minor tissues in response to CuO NPs exposure. Moreover, chloroplasts were confirmed as a site of ROS production. These findings are helpful for better understanding the biological responses of aquatic plants upon NPs exposure.