Rice (Oryza sativa L.), a major staple food for billions of people, was assessed for its phytotoxicity of copper oxide nanoparticle (CuO NPs, size < 50 nm). Under hydroponic condition, seven days of exposure to 62.5, 125, and 250 mg/L CuO NPs significantly suppressed the growth rate of rice seedlings compared to both the control and the treatment of supernatant from 250 mg/L CuO NP suspensions. In addition, physiological indexes associated with antioxidants, including membrane damage and antioxidant enzyme activity, were also detected. Treatment with 250 mg/L CuO NPs significantly increased malondialdehyde (MDA) content and electrical conductivity of rice shoots by 83.4% and 67.0%, respectively. The activity of both catalase and superoxide dismutase decreased in rice leaves treated with CuO NPs at the concentration of 250 mg/L, while the activity of the superoxide dismutase significantly increased by 1.66 times in rice roots exposed to 125 mg/L CuO NPs. The chlorophyll, including chlorophyll a and chlorophyll b, and carotenoid content in rice leaves decreased with CuO NP exposure. Finally, to explain potential molecular mechanisms of chlorophyll variations, the expression of four related genes, namely, Magnesium chelatase D subunit, Chlorophyll synthase, Magnesium-protoporphyrin IX methyltransferase, and Chlorophyllide a oxygenase, were quantified by qRT-PCR. Overall, CuO NPs, especially at 250 mg/L concentration, could affect the growth and development of rice seedlings, probably through oxidative damage and disturbance of chlorophyll and carotenoid synthesis.