Nanoparticles (NPs) can be used in several ways in agriculture, including increasing production rates and improving nutritional values in plants. The present study aims to clarify how biogenic copper oxide nanoparticles (CuO NPs) applied by two routes of exposure (foliar spray and soil irrigation) affect the elemental uptake by lettuce. In vivo experiments using lettuce (n = 4) were performed with CuO NPs in comparison with copper salt (CuSO4), considering a final mass added of 20 mg of CuO per plant. The elemental composition of roots was mostly affected by the soil irrigation exposure for both Cu forms (NPs and salt). Neither Cu form added by soil irrigation was translocated to leaves. Copper concentration in leaves was mainly affected by foliar spray exposure for both Cu forms (NPs and salt). All Cu forms through foliar spray were sequestered in the leaves and no translocation to roots was observed. Foliar spray of CuO NPs caused no visual damage in leaves, resulted in less disturbance of elemental composition, and improved dry weight, number of leaves, CO2 assimilation, and the levels of K, Na, S, Ag, Cd, Cr, Cu, and Zn in leaves without causing significant changes in daily intake of most elements, except for Cu. Although Cu concentration increased in leaves by foliar spray of CuO NPs, it remained safe for consumption.Funda????o de Amparo ?? Pesquisa do Estado de S??o Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico (CNPq)Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior (CAPES)FAPESP: 17/05029-8; 17/20914-8; 18/08194-2; 19/26337-8; 16/10060-9CNPq: 429555/2018-0; 153204/2018-4; 313117/2019-5; 404815/2018-9CAPES: 88887.363169/2019-00; 001