Understanding the complex mechanisms involved in plant response to nanoparticles (NPs) is indispensable in assessing the environmental impact of nano-pollutants. Plant leaves can directly intercept or absorb NPs deposited on their surface; however, the toxicity mechanisms of NPs to plant leaves are unclear. In this study, lettuce leaves were exposed to copper oxide nanoparticles (CuO-NPs, 0, 100, and 1000 mg/L) for 15 days, then physiological tests and transcriptomic analyses were conducted to evaluate the negative impacts of CuO-NPs. Both physiological and transcriptomic results demonstrated that CuO-NPs adversely affected plant growth, photosynthesis, and enhanced reactive oxygen species (ROS) accumulation and antioxidant system activity. The comparative transcriptome analysis showed that 2270 and 4264 genes were differentially expressed upon exposure to 100 and 1000 mg/L CuO-NPs. Gene expression analysis suggested the ATP-binding cassette (ABC) transporter family, heavy metal-associated isoprenylated plant proteins (HIPPs), endocytosis, and other metal ion binding proteins or channels play significant roles in CuO-NP accumulation by plant leaves. Furthermore, the variation in antioxidant enzyme transcript levels (POD1, MDAR4, APX2, FSDs), flavonoid content, cell wall structure and components, and hormone (auxin) could be essential in regulating CuO-NPs-induced stress. These findings could help understand the toxicity mechanisms of metal NPs on crops, especially NPs resulting from foliar exposure.