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Biomolecular coatings (coronas) that form on nanomaterials have been widely investigated in animal and bacterial cell culture and in the extracellular and intracellular fluids of animals. Such coronas influence the distribution of nanoparticles within organisms, their uptake by cells, and their storage in intracellular compartments. Plants can be exposed to nanoparticles via either intentional application of nanomaterials in agriculture or inadvertently due, for example, to biosolids amendment of soils. Development of a mechanistic understanding of nanoparticle transport and fate within plants requires consideration of corona acquisition within plants, particularly within the vascular fluids that transport nanoparticles throughout plants. Here, we examine the interactions between copper oxide (CuO) nanoparticles and pumpkin xylem fluid to understand corona formation in an important part of the plant vasculature system. We used CuO nanoparticles because they have emerged as a promising micronutrient source for the suppression of fungal diseases. The corona was composed primarily of proteins, despite the higher abundance of carbohydrates in xylem fluid. We used X-ray photoelectron spectroscopy to determine the thickness of the protein corona. Polyacrylamide gel electrophoresis revealed that protein binding to the CuO nanoparticle surface was selective; the most abundant proteins in the corona were not the most abundant ones in the xylem fluid. We used in situ attenuated total reflectance Fourier-transform infrared spectroscopy to show that the protein–CuO NP interactions were quasi-irreversible, while carbohydrate–CuO interactions were reversible. Corona formation is expected to influence the distribution and transformation of nanomaterials in plants.